Lilrb1-based chimeric antigen receptor

ABSTRACT

Provided are chimeric antigen receptors having the hinge, transmembrane region, and/or intracellular domain of LILRB1, or functional fragments or variants thereof. Also provided herein are cells comprising the LILRB1 based receptors, and methods of making and using same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.17/230,637, filed Apr. 14, 2021, which is a continuation ofInternational Application No. PCT/US2020/064607, filed on Dec. 11, 2020,which claims priority to and benefit of U.S. Provisional PatentApplication Nos. 63/085,969 filed on Sep. 30, 2020, and 62/946,888,filed on Dec. 11, 2019, the contents of each of which are incorporatedherein by reference in their entireties.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted inASCII format via EFS-WEB and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jan. 31, 2022 is namedA2BI_015_06US_SeqList_ST25.txt and is 228 KB in size.

BACKGROUND

Chimeric antigen receptor (CAR) T cell therapy, and T Cell Receptor(TCR) therapy, is proving to be an effective therapeutic approach tovarious diseases, particularly hematological malignancies but also othercancers. CAR NK cells may also have clinical applications. ConventionalCARs provide a stimulatory signal to the engineered immune cell (e.g. aT cell or an NK cell). In CAR-T cells, this results in killing activitytowards the target cell identified by the antigen-binding domain of theCAR. Inhibitory CARs (iCARs) have been developed as a means to controlcell activity or restrict the activity of an activator CAR to specificcell types. Fedorov et al. Sci. Transl. Med. 5(215):215ra172 (2013). Theinhibitory CAR generally has the intracellular domain of an inhibitorysignaling molecule (such as PD-1 or CTLA-4) fused to an antigen-bindingdomain (e.g., a single-chain variable fragment, scFv) through atransmembrane region and optionally a hinge region.

Numerous alternative iCAR architectures have been described in the art.However, there remains an unmet need for novel alternative inhibitoryreceptors and identification of particular inhibitory receptorarchitectures having superior performance, along with associatedcompositions and methods of use thereof.

SUMMARY

In one aspect, the disclosure provides chimeric antigen receptors havingthe hinge, transmembrane region, and/or intracellular domain of LILRB1,or functional fragments or variants thereof. The chimeric antigenreceptor may include single polypeptide, or more than one polypeptide.The receptors may include one or more, or all of the following: (a) anLILRB1 hinge domain or functional fragment or variant thereof; (b) anLILRB1 transmembrane domain or a functional variant thereof; and (c) anLILRB1 intracellular domain or a functional variant thereof, such as anLILRB1 intracellular domain and/or an intracellular domain comprising atleast one immunoreceptor tyrosine-based inhibitory motif (ITIM) found inthe polypeptide sequence of LILRB1. In some embodiments, the receptorcomprises at least two ITIMS found in the polypeptide sequence ofLILRB1. The ITIMs of LILRB1 are NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ IDNO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). The receptormay include one, two, three, four, five, six, or more of these ITIMs, inany combination including multiple copies of the same ITIM.

In some embodiments of the receptors of the disclosure, theintracellular domain comprises both ITIMs NLYAAV (SEQ ID NO: 8) andVTYAEV (SEQ ID NO: 9). In some embodiments, the intracellular domaincomprises a sequence at least 95% identical to SEQ ID NO: 12. In someembodiments, the intracellular domain comprises both ITIMs VTYAEV (SEQID NO: 9) and VTYAQL (SEQ ID NO: 10). In some embodiments, theintracellular domain comprises a sequence at least 95% identical to SEQID NO: 13. In some embodiments, the intracellular domain comprises bothITIMs VTYAQL (SEQ ID NO: 10) and SIYATL (SEQ ID NO: 11). In someembodiments, the intracellular domain comprises a sequence at least 95%identical to SEQ ID NO: 14. In some embodiments, the polypeptidecomprises an intracellular domain comprising at least threeimmunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein eachITIM is independently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). In someembodiments, the intracellular domain comprises the ITIMs NLYAAV (SEQ IDNO: 8), VTYAEV (SEQ ID NO: 9), and VTYAQL (SEQ ID NO: 10). In someembodiments, the intracellular domain comprises a sequence at least 95%identical to SEQ ID NO: 15. In some embodiments, the intracellulardomain comprises the ITIMs VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO: 11). In some embodiments, the intracellulardomain comprises a sequence at least 95% identical to SEQ ID NO: 16. Insome embodiments, the intracellular domain comprises the ITIMs NLYAAV(SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), andSIYATL (SEQ ID NO: 11). In some embodiments, the intracellular domaincomprises a sequence at least 95% identical to SEQ ID NO: 17. In someembodiments, the intracellular domain comprises a sequence at least 95%identical to the LILRB1 intracellular domain (SEQ ID NO: 7). In someembodiments, the intracellular domain comprises a sequence of SEQ IDNOS: 12-17.

In some embodiments of the receptors of the disclosure, the polypeptidecomprises the LILRB1 transmembrane domain or a functional variantthereof. In some embodiments, the LILRB1 transmembrane domain or afunctional variant thereof comprises a sequence at least 95% identicalto SEQ ID NO: 5. In some embodiments, the LILRB1 transmembrane domaincomprises SEQ ID NO: 5.

In some embodiments of the receptors of the disclosure, the polypeptidecomprises the LILRB1 hinge domain or functional fragment or variantthereof. In some embodiments, the LILRB1 hinge domain or functionalfragment or variant thereof comprises a sequence at least 95% identicalto SEQ ID NO: 4, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 80, SEQ ID NO:81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84 or SEQ ID NO: 93. Insome embodiments, the LILRB1 hinge domain comprises a sequence identicalto SEQ ID NO: 4, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 80, SEQ ID NO:81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84 or SEQ ID NO: 93. Insome embodiments, the LILRB1 hinge domain or functional fragment orvariant thereof comprises a sequence at least 95% identical to SEQ IDNO: 4, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 80, SEQ ID NO:81, SEQ IDNO: 82, SEQ ID NO: 83 or SEQ ID NO: 84. In some embodiments, the LILRB1hinge domain comprises a sequence identical to SEQ ID NO: 4, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ IDNO: 83 or SEQ ID NO: 84.

In some embodiments of the receptors of the disclosure, the polypeptidecomprises: (a) an LILRB1 hinge domain or functional fragment or variantthereof, and (b) the LILRB1 transmembrane domain or a functional variantthereof. In some embodiments, the polypeptide comprises a sequence atleast 95% identical to SEQ ID NO: 20.

In some embodiments of the receptors of the disclosure, the polypeptidecomprises: (a) the LILRB1 transmembrane domain or a functional variantthereof, and (b) an LILRB1 intracellular domain and/or an intracellulardomain comprising at least two immunoreceptor tyrosine-based inhibitorymotifs (ITIMs), wherein each ITIM is independently selected from NLYAAV(SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), andSIYATL (SEQ ID NO: 11). In some embodiments, the polypeptide comprises asequence at least 95% identical to SEQ ID NO: 21. In some embodiments,the polypeptide comprises a sequence of SEQ ID NO. 21.

In some embodiments of the receptors of the disclosure, the polypeptidecomprises: (a) an LILRB1 hinge domain or functional fragment or variantthereof, (b) an LILRB1 transmembrane domain or a functional variantthereof; and (c) an LILRB1 intracellular domain and/or an intracellulardomain comprising at least two immunoreceptor tyrosine-based inhibitorymotifs (ITIMs), wherein each ITIM is independently selected from NLYAAV(SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), andSIYATL (SEQ ID NO: 11).

In some embodiments of the receptors of the disclosure, the polypeptidecomprises a sequence at least 95% identical to SEQ ID NO: 2 or SEQ IDNO: 3. In some embodiments, the polypeptide comprises a sequence atleast 99% identical to SEQ ID NO: 20. In some embodiments, thepolypeptide comprises a sequence at least 99% identical to SEQ ID NO:21. In some embodiments, the polypeptide comprises a sequence at least99% identical to SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, thepolypeptide comprises a sequence identical to SEQ ID NO: 20. In someembodiments, the polypeptide comprises a sequence identical to SEQ IDNO: 21. In some embodiments, the polypeptide comprises a sequenceidentical to SEQ ID NO: 2 or SEQ ID NO: 3.

In some embodiments of the receptors of the disclosure, the polypeptidecomprises antigen-binding domain. In some embodiments, theantigen-binding domain is an antigen-binding domain other than theLILRB1 extracellular ligand binding protein. In some embodiments, thepolypeptide comprises two or more antigen-binding domains. In someembodiments, the antigen-binding domain comprises a single chainvariable fragment (scFv). In some embodiments, the receptor comprises asecond polypeptide. In some embodiments, the first polypeptide comprisesa first chain of an antibody and the second polypeptide comprise asecond chain of said antibody. In some embodiments, the receptorcomprises a Fab fragment of an antibody. In some embodiments, (a) thefirst polypeptide comprises an antigen-binding fragment of the heavychain of the antibody, and (b) the second polypeptide comprises anantigen-binding fragment of the light chain of the antibody. In someembodiments, (a) the first polypeptide comprises an antigen-bindingfragment of the light chain of the antibody, and (b) the secondpolypeptide comprises an antigen-binding fragment of the heavy chain ofthe antibody. In some embodiments, the first polypeptide comprises afirst chain of a T-cell receptor (TCR) and the second polypeptidecomprises a second chain of said TCR. In some embodiments, in thereceptor comprises an extracellular fragment of a T cell receptor (TCR).In some embodiments, (a) the first polypeptide comprises anantigen-binding fragment of an alpha chain of the TCR, and (b) thesecond polypeptide comprises an antigen-binding fragment of the betachain of the TCR. In some embodiments, (a) the first polypeptidecomprises an antigen-binding fragment of the beta chain of the TCR, and(b) the second polypeptide comprises an antigen-binding fragment of thealpha chain of the TCR. In some embodiments, the receptor comprises asingle-chain TCR. In some embodiments, the scFv comprises thecomplementarity determined regions (CDRs) of any one of SEQ ID NOS:22-33. In some embodiments, the scFv comprises a sequence at least 95%identical to any one of SEQ ID NOS: 35-46 or 125. In some embodiments,the scFv comprises a sequence at least 95% identical to any one of SEQID NOS: 35, 39, 46 or 125. In some embodiments, the scFv comprises asequence identical to any one of SEQ ID NOS: 35-46 or 125. In someembodiments, the scFv comprises a sequence identical to any one of SEQID NOS: 35, 39, 46 or 125. In some embodiments, the heavy chain of theantibody comprises the heavy chain CDRs of any one of SEQ ID NOS: 25-27or 31-33, and wherein the light chain of the antibody comprises thelight chain CDRs of any one of SEQ ID NOS: 22-24 or 28-30. In someembodiments, the heavy chain of the antibody comprises a sequence atleast 95% identical to the heavy chain portion of any one of SEQ ID NOS:35-46 or 125, and wherein the light chain of the antibody comprises asequence at least 95% identical to the light chain portion of any one ofSEQ ID NOS: 35-46 or 125. In some embodiments, the heavy chain of theantibody comprises a sequence identical to the heavy chain portion ofany one of SEQ ID NOS: 35-46 or 125, and wherein the light chain of theantibody comprises a sequence identical to the light chain portion ofany one of SEQ ID NOS: 35-46 or 125. In some embodiments, the heavychain of the antibody comprises a sequence identical to the heavy chainportion of any one of SEQ ID NOS: 35, 39, 46 or 125, and wherein thelight chain of the antibody comprises a sequence identical to the lightchain portion of any one of SEQ ID NOS: 35, 39, 46 or 125.

In some embodiments of the receptors of the disclosure, the receptorcomprises an amino acid sequence at least 95% identical to any one ofSEQ ID NOS: 47-71, 77-79, 89-92, 120 or 122. In some embodiments, thereceptor comprises an amino acid sequence of SEQ ID NOS: 47-71, 77-79,89-92, 120 or 122.

In some embodiments of the receptors of the disclosure, the receptor isan inhibitory receptor.

The disclosure provides a polynucleotide comprising a nucleic acidsequence encoding the receptor or polypeptide of the disclosure.

The disclosure provides a vector comprising the polynucleotide of thedisclosure. In some embodiments, the vector further comprises a sequenceencoding a promoter operably linked to the polynucleotide.

The disclosure provides an immune cell comprising the receptor,polynucleotide, polypeptide or receptor of the disclosure. In someembodiments, the immune cell activation is reduced when the cell iscontacted with the antigen or a cell expressing the antigen on itssurface. In some embodiments, immune cell activation comprisesexpression of a gene operatively linked to an NFAT promoter. In someembodiments, the immune cell is a T cell. In some embodiments, furthercomprises an activator receptor. In some embodiments, the activatorreceptor is a chimeric antigen receptor or a T cell receptor.

The disclosure provides methods making an immune cell, comprisingintroducing the polynucleotide or vector of the disclosure into theimmune cell. In some embodiments, the immune cell expresses thereceptor. In some embodiments, the cell is an immune cell. In someembodiments, the immune cell is a T cell. In some embodiments, immunecell activation is reduced when the cell is contacted with an antigenspecific to the chimeric antigen receptor, or a cell expressing theantigen on its surface. In some embodiments, immune cell activationcomprises expression of a gene operatively linked to an NFAT promoter.

The disclosure provides methods of treating a subject with a disease ora disorder, comprising administering to the subject a plurality of theimmune cells of the disclosure. In some embodiments, the disease ordisorder is cancer.

The disclosure provides a kit, comprising the receptor, polypeptide,polynucleotide, vector or immune cell of the disclosure.

The disclosure provides an immune cell comprising a chimeric antigenreceptor comprising a polypeptide, wherein the polypeptide sequenceshares at least 95% identity or at least 100% identity to SEQ ID NO: 21.

In some embodiments of the immune cells of the disclosure, thepolypeptide sequence shares at least 95% identity or at least 100%identity to SEQ ID NO: 3. In some embodiments, the polypeptide sequenceshares at least 95% identity or at least 100% identity to SEQ ID NO: 2.In some embodiments, the chimeric antigen receptor comprises anantigen-binding domain comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1,CDR-H2, CDR-H3 sequences according to SEQ ID NO: 22-27, respectively. Insome embodiments, the chimeric antigen receptor comprises anantigen-binding domain comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1,CDR-H2, CDR-H3 sequences according to SEQ ID NO: 28-33, respectively. Insome embodiments, the polypeptide sequence shares at least 95% identityor at least 100% identity to SEQ ID NO: 122. In some embodiments, thepolypeptide sequence shares at least 95% identity or at least 100/6identity with any one of SEQ ID NOS: 35, 39, 46 or 125 in combinationwith SEQ ID NO: 2.

In some embodiments, the immune cell is a T cell. In some embodiments,the T cell comprises a chimeric antigen receptor or T cell receptor thatspecifically binds to a target expressed on tumor cells. In someembodiments, the T cell comprises a chimeric antigen receptor or T cellreceptor that specifically binds to a target selected from etiolatereceptor, αvββ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22,CD30, CD33, CD37, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138, CD171,CEA, DLL4, EGP-2, EGP-40, CSPG4, EGFR, EGFR family including ErbB2(HER2), EGFRvIII, EPCAM, EphA2, EpCAM, FAP, FBP, fetal acetylcholinereceptor, Fzd7, GD2, GD3, Glypican-3 (GPC3), h5T4, IL-11R, IL13R-a2,KDR, κ light chain, λ light chain, LeY, LI CAM, MAGE-A1, mesothelin, MHCpresented peptides, MUC1, MUC16, NCAM, NKG2D ligands, Notch1, Notch2/3,NY-ESO-1, PRAME, PSCA, PSMA, Survivin, TAG-72, TEMs, TERT, VEGFR2, andROR1.

The disclosure provide methods of treating and/or preventing cancer in asubject in need thereof, comprising administering to the subject theimmune cells of the disclosure. In some embodiments, the methodcomprises treating and/or preventing cancer in a subject in needthereof, comprising administering to the subject the immune cells of thedisclosure.

Illustrative CARs provided herein include, without limitation,antibody-based CARs such as single-chain variable fragment (scFv) CARs,Fab CARs, or others; and T cell receptor (TCR)-based CARs.

In other aspects, the disclosure provides polynucleotides encoding suchreceptors; vectors for delivery of such polynucleotides; and immunecells with such polynucleotide and receptors.

In further aspects, the disclosure provides methods of introducingpolynucleotide or vectors encoding such receptors into a cell.Advantageously, immune cell activation is reduced when the cell iscontacted with the antigen or a cell expressing the antigen on itssurface.

Yet further aspects and embodiments of the invention are provided in thedetailed description that follows.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an illustrative diagram of domain arrangement in anembodiment having a ligand binding domain (LBD), hinge, transmembrane(TM), and intracellular signaling domain (ICD).

FIGS. 2A-2B show illustrative diagrams of domain arrangements inembodiments having a ligand binding domain (LBD), hinge, transmembrane(TM), and intracellular signaling domain (ICD). When the ligand bindingdomain comprises two peptides, for example a heterodimeric LDB from a TCell Receptor, each peptide may be fused with a hinge, TM andintracellular domain (FIG. 2A). Alternatively, only one peptide of theligand binding domain may be fused to the hinge, TM and intracellulardomain (FIG. 2B).

FIG. 3 shows four illustrative embodiments of immune cells having anactivator scFv-based chimeric antigen receptor (CAR) [101 and 102] oractivator T cell receptor [103 and 104] and an inhibitory scFv based CAR[101 and 103] or inhibitory TCR-based CAR [102 and 104].

FIG. 4 shows a chart of luminescence in an NFAT-based reporter assay(relative luminescence units, RLU) at varying concentrations of MAGE-A3activating peptide 1 (MP1, microliters, μM) for the indicated constructsin the presence of 50 μM NY-ESO-1 peptide.

FIG. 5 shows a chart of luminescence in an NFAT-based reporter assay(RLU) at varying concentrations of MAGE-A3 peptide 1 (MP1, nM) for theindicated constructs in the presence of various concentrations (μM) ofNY-ESO-1 peptide.

FIG. 6 shows a chart of luminescence in an NFAT-based reporter assay(RLU) at varying concentrations of MAGE-A3 peptide 1 (MP1, nM) for theindicated constructs in the presence of various concentrations (μM) ofNY-ESO-1 peptide.

FIG. 7 shows a chart of luminescence in an NFAT-based reporter assay(RLU) at varying concentrations of MAGE-A3 peptide 1 (MP1, nM) for theindicated constructs in the presence of 50 μM NY-ESO-1 peptide.

FIG. 8 shows a chart of luminescence in an NFAT-based reporter assay(RLU) at varying concentrations of MAGE-A3 peptide 1 (MP1, nM) for theindicated constructs in the presence of 50 μM NY-ESO-1 peptide.

FIG. 9 shows a chart of luminescence in an NFAT-based reporter assay(RLU) at varying concentrations of MAGE-A3 peptide 1 (MP1, nM) for theindicated constructs in the presence of 5 μM NY-ESO-1 peptide.

FIG. 10 shows a chart of luminescence in an NFAT-based reporter assay(RLU) at varying concentrations of MAGE-A3 peptide 1 (MP1, nM) for theindicated constructs in the presence of 50 μM NY-ESO-1 peptide.

FIG. 11 shows a chart of luminescence in an NFAT-based report assay(RLU) at varying concentrations of MAGE-A3 peptide 1 (MP1, nM) for theindicated constructs in the presence of 50 μM NY-ESO-1 peptide.

FIG. 12A is a plot showing the effect of LIR-1 hinge on the ability ofan HLA-A*02 scFv inhibitory receptor to block activation of Jurkat cellsby a KRAS TCR. H: hinge, T: transmembrane domain, ICD: intracellulardomain, s: short. LIR-1 constructs are described in more detail in FIG.12B. Humanized PA2.1 and humanized BB7.2 with shorter LIR-1 hinge blocksimilarly to original, longer hinge.

FIG. 12B is a plot and a table showing EC50 shift (+/−HLA-A*02 targetcells) for Jurkat cells expressing a KRAS TCR activator and the HLA-A*02scFv LIR-1 inhibitory receptor shown in the table at bottom.

FIG. 13A is a plot showing the effect of LIR-1 hinge on the ability ofan HLA-A*02 inhibitory receptor to block activation of Jurkat cells by aKRAS TCR. H: hinge, TM: transmembrane domain, ICD: intracellular domain,s: short; tr: truncated. LIR-1 constructs are described in more detailin FIG. 13B. Mouse PA2.1 with slightly longer hinges function similarlyto original LIR-1 hinge in T2-Jurkat assay.

FIG. 13B is a plot and a pair of tables showing EC50 shift (+/−HLA-A*02target cells) for Jurkat cells expressing a KRAS TCR activator and theHLA-A*02 scFv LIR-1 inhibitory receptors shown in the table at bottom,with hinge lengths shown in the table at left.

FIG. 14A is a diagram showing a schematic of T2-Jurkat experiments toevaluate blocker constructs.

FIG. 14B is a plot, table and diagram showing the effect of variousNY-ESO-1 scFv LBD blocker modules (PD-1, CTLA-4, LIR-1) on EC50 ofMAGE-A3 CAR activator (MP1-LBD 1-CAR), measured by MAGE peptidetitration of cells loaded with a fixed (50 μM) NY-ESO-1 blocker peptideconcentration. In each of FIGS. 14B-14F, NFAT-luciferase signal ofJurkat cells transfected with either activator CAR alone or incombination with each blocker receptor after 6 hours of co-culture withactivator and blocker peptide-loaded T2 cells was assayed. The baseline(Jurkat only) varies with different activator alone constructs and canbe especially high with CARs; in most cases expression of the blockerreceptor absent its ligand suppresses the baseline. Activator peptideconcentrations range from 0 then 10⁻⁶ to 10² μM and luminescencemeasurements ranged from 0 to 80000 RLU.

FIG. 14C is a plot, table and diagram showing the effect of LIR-1blocker receptor with various scFv LBDs (ESO, MP1 LBD 1, MP1 LBD 2, HPVE6 LBD 1, HPV E6 LBD 2, HPV E7) on EC50 of MAGE-A3 CAR activator(MP1-CAR) when loaded with corresponding blocker peptide at fixed (50μM) peptide concentration, as in FIG. 14B. RLU=relative light units;error bars indicate ±SD (n=2). Activator peptide concentrations rangefrom 0 then 10⁻³ to 10² μM and luminescence measurements ranged from 0to 100000 RLU.

FIG. 14D is a plot, table and diagram showing the effect of LIR-1blocker receptor with NY-ESO-1 scFv LBD on EC50 of different MAGE-A3 CARactivators (MP1-LBD 1-CAR or MP2-CAR) when loaded with 50 uM NY-ESO-1blocker peptide. Activator peptide concentrations range from 0 then 10⁻⁴to 10² μM and luminescence measurements ranged from 0 to 80000 RLU.

FIG. 14E is a plot, table and diagram showing the effect of LIR-1blocker receptor with NY-ESO-1 scFv LBD on EC50 of different TCRactivators (MP1-TCR, MP2-TCR, HPV E6-TCR) when loaded with 50 uMNY-ESO-1 blocker peptide. Activator peptide concentrations range from 0then 10⁻⁴ to 10² μM and normalized luminescence measurements ranged from0 to 150 RLU.

FIG. 14F is a plot, table and diagram showing the effect of LIR-1blocker receptor with NY-ESO-1 TCR LBDs on EC50 of MAGE-A3 CAR and TCRactivators (MP1-LBD 1-CAR, MP1-TCR) when loaded with 50 uM NY-ESO-1blocker peptide. RLU=relative light units; error bars indicate ±SD(n=2). Activator peptide concentrations range from 0 then 10⁻⁷ to 10¹ μMand normalized luminescence measurements ranged from 0 to 150 RLU.

FIG. 15 is a plot showing the effect of blocker peptide loading (50 μMeach of NY-ESO-1, MAGE-A3, HPV E6, and HPV E7) on activating MAGE-A3CAR. MP2-CAR [0 μM], EC50=44 nM; MP2-CAR [50 μM HPVp2], EC50=495 nM.RLU=relative light units; error bars indicate ±SD (n=2).

FIG. 16A is a series of plots showing NFAT-luciferase signal of Jurkatcells transfected with either activator MAGE-A3 CAR alone or incombination with various amounts of NY-ESO-1 scFv LBD blocker (DNAratios of activator and blocker receptors components shown on left asA:B, activator receptor:blocker receptor after 6 h of co-culture withactivator and blocker peptide-loaded T2 cells. T2 cells were loaded withtitrated amounts of activator MAGE-A3 peptide and a fixed amount ofblocker NY-ESO-1 peptide concentration. Activator and/or blocker peptideconcentrations range from 0 then 10⁻⁶ to 10² μM and luminescencemeasurements ranged from 0 to 200000 RLU.

FIG. 16B is a series of plots showing NFAT-luciferase signal of Jurkatcells transfected with either activator MAGE-A3 CAR alone or incombination with various amounts of NY-ESO-1 scFv LBD blocker. T2 cellswere loaded with titrated amounts of blocker NY-ESO-1 peptide and afixed amount of activator MAGE-A3 peptide concentration above the Emaxconcentration (˜0.1 mM). Activator and/or blocker peptide concentrationsrange from 10⁻⁵ to 10² μM and luminescence measurements ranged from 0 to200000 RLU.

FIG. 16C is a series of plots and two tables showing NFAT-luciferasesignal of Jurkat cells transfected with either activator MAGE-A3 CARalone or in combination with various amounts of NY-ESO-1 scFv LBDblocker. The x-value blocker NY-ESO-1 peptide concentrations from FIG.16B were normalized to the constant activator MAGE peptideconcentrations used for each curve and plotted on the x-axis. The ratioof blocker peptide to activator peptide required for 50% blocking (IC50)are indicated for each curve. For all DNA ratios, the B:A peptide ratiorequired is less than 1 indicating that, for this pair of activator CARand blocker, similar (or fewer) blocker pMHC antigens are required ontarget cells to block activator pMHC antigens. Activator and/or blockerpeptide concentrations range from 10⁻¹⁰ to 10⁴ μM and luminescencemeasurements ranged from 0 to 200000 RLU.

FIG. 16D is a table and a plot showing that blocking CD19-CAR activatoris possible with pMHC blockers at blocker pMHC antigen densities similarto those required to activate pMHC CARs. NFAT-luciferase signal ofJurkat cells transfected with either activator CD19 CAR alone or incombination with various amounts of NY-ESO-1 blocker (DNA ratios shown)after 6 h of co-culture with blocker peptide-loaded T2 cells whichexpress endogenous levels of CD19 antigen. The IC50 is estimated fromthe inhibition curves to range from 0.1-1.0 mM, corresponding to˜1,500-3,500 pMHCs/cell. RLU=relative light units; error bars indicate±SD (n=2).

FIG. 17A is a plot showing the effect of NY-ESO-1-LIR-1 blocker on EC50of activating MAGE-A3 CAR (MP1-CAR) when loaded with variousconcentrations of NY-ESO-1 blocker peptide. The EC50 shifts are greateras the concentration of blocker peptide (NY-ESO-1) increases. The shiftin the presence of a negative-control HPV peptide (binds HLA-A*02 butnot NY-ESO-1 blocker scFv) is routinely seen and believed to be causedby competition of the control peptide for binding sites on the T2HLA-A*02 molecules, reducing the number of activator targets. Activatorconcentrations range from 0 then 10⁴ to 10² μM and luminescencemeasurements ranged from 0 to 140000 RLU.

FIG. 17B is a plot showing the effect of modified LIR-1 blockerreceptors containing no ICD or a mutated ICD with NY-ESO-1 scFv LBD onEC50 of MAGE-A3 CAR activator (MP2-CAR) when loaded with 10 μM ofNY-ESO-1 blocker peptide. Activator concentrations range from 0 then 10¹to 10² μM and luminescence measurements ranged from 0 to 140000 RLU.

FIGS. 17C-17E are a series of plots showing the effect of variousNY-ESO-1 scFv LBD blocker receptors (CTLA-4 (FIG. 17C), PD-1 (FIG. 17D)and LIR-1 (FIG. 17E)) on EC50 of MAGE-A3 CAR activator (MP1-LBD 1-CAR)when blockers are stimulated or unstimulated. NFAT-luciferase signal ofJurkat cells transfected with either activator CAR alone or incombination with each blocker after 6 h of co-culture withpeptide-loaded T2 cells. T2 cells were loaded with titrated amounts ofactivating MAGE peptide and tested with and without loading additionalconstant amount (50 μM) of NY-ESO-1 blocker peptide. RLU=relative lightunits; error bars indicate ±SD (n=2). Activator concentrations rangefrom 0 then 10⁻⁶ to 10² μM and luminescence measurements ranged from 0to 100000 RLU.

FIG. 18A is a diagram and a pair of plots showing that Jurkat cellstransfected with either HPV E7-CAR or HPV E7-CAR & A2-LIR-1 co-culturedwith beads displaying various ratios of activator (HPV E7) and blocker(NY-ESO-1) antigen demonstrates blocking in cis but not trans.

FIG. 18B is a plot showing that HLA-A*02-LIR-1 blocker receptor blocksCD19-CAR activator at various activator to blocker ratios. Ratios rangedfrom 0 to 10 and luminescence (RLU) ranged from 0 to 70000.

FIG. 18C is a plot showing surface expression of titrated HLA-A*02 (A2)LIR-1 blocker receptor.

FIG. 18D is a plot showing that an scFv against HLA-A*02 can also serveas an activator when fused to activator CAR. T2 cells expressingendogenous HLA-A*02 serve as the target. RLU=relative light units; errorbars indicate f SD (n=2).

FIG. 19A is a plot showing the effect of LIR-1 blocker receptor withNY-ESO-1 scFv LBD on EC50 of different TCR activators (MP1-TCR, MP2-TCR,HPV E6-TCR) when loaded with NY-ESO-1 blocker peptide. For FIG. 14E eachset was normalized to the Emax of the curve showing response of theactivator only. Activator concentrations range from 0 then 10⁻⁴ to 10²μM and luminescence measurements ranged from 0 to 140000 RLU.

FIG. 19B is a plot showing the effect of LIR-1 blocker receptor withNY-ESO-1 TCR LBDs on EC50 of MAGE-A3 CAR and TCR activators (MP1-LBD1-CAR, MP1-TCR). Each set was normalized to the Emax of the curveshowing response of the activator only. RLU=relative light units; errorbars indicate ±SD (n=2). Activator concentrations range from 0 then 10⁻⁷to 10¹ μM and luminescence measurements ranged from 0 to 140000 RLU.

FIG. 20A is a plot showing primary T cells (donor 1) transduced with HPVE7-TCR activator and ESO-LIR-1 blocker shifts EC50˜25× in primary T cellkilling assay (HPV E7 TCR EC50=0.044 nM; HPV E7 TCR+ESO-LIR-1, EC50=1.1nM). The assay was performed using peptide-loaded MCF7 target cells at a3:1 E:T. Luciferase measurement represents live target cells at 48hours.

FIG. 20B is a plot showing that HLA-A*02-LIR-1 blocks NY-ESO-1 CARactivator at various activator:blocker DNA ratios in Jurkat cells usingNY-ESO-1 peptide-loaded T2 target cells. RLU=relative light units; errorbars indicate ±SD (n=2).

FIG. 21A is a series of images and plots showing that primary T cells(donor 1) transduced with CD19 CAR activator and HLA-A*02 blockerdistinguish “tumor” cells from “normal” cells in in vitro cytotoxicityassay and demonstrate selective killing of “tumor” cells in mixed targetcell assay at 3:1 E:T. Images shown were captured at 72 hours. Shown areUntransduced, CD19-CAR T cells, and CD19-CAR T+A2-LIR-1. Measurementswere taken between 0 and 150 hours and normalized fluorescent proteinintensity (GFP or RFP) ranged from 0 to 10.

FIG. 21B is a series of plots showing that primary T cells (donor 1)selectively kill tumor cells similarly at various tumor to “normal” cellratios at 3:1 E:T ratios in Incucyte imaging assay. RLU values werenormalized against target cell mixtures grown in the absence of primaryT cells. Shown are Untransduced, CD19-CAR T cells, and CD19-CART+A2-LIR-1. Measurements were taken between 0 and 150 hours andnormalized fluorescent protein intensity (GFP or RFP) ranged from, onthe top row, from left to right: 0 to 3×10⁷, 0 to 2×10⁷, 0 to 1.6×10⁷, 0to 7×10⁶, 0 to 2×10⁶; bottom row, from left to right: 0 to 7×10⁵, 0 to2×10⁵, 0 to 4×10⁵, 0 to 6×10⁵, 0 to 7×10⁵.

FIG. 21C is a series of plots showing that primary T cells (donor 1)selectively kill tumor cells similarly at various tumor to “normal” cellratios at 3:1 E:T ratios in quantitative target cell lysis and IFNγsecretion. Shown are Untransduced, CD19-CAR T cells, and CD19-CART+A2-LIR-1.

FIG. 22A is a pair of plots showing that Jurkat cells transfected withMSLN LBD1-CAR or MSLN LBD1-CAR & A2-LIR-1 co-cultured with K562 cellsexpressing either MSLN or MSLN & HLA-A*02 shows blocking of activationby a high-density antigen with A2-LIR-1 blocker only in the presence ofHLA-A*02.

FIG. 22B is a pair of plots showing that killing of endogenous MSLN+HeLa cells by MLSN LBD1-CAR T cells is blocked in the presence ofHLA-A*02 with the A2-LIR-1 blocker.

FIG. 22C is a pair of plots showing killing of endogenous MSLN+ HeLacells by MLSN LBD2-CAR T cells. The effect of A2-LIR-1 blocker on T cellkilling is in part controlled by the activator LBD.

FIG. 23 is a plot showing that LIR-1 blocker receptors have littleeffect on killing efficacy of activator in absence of blocker antigen.In the absence of NY-ESO-1 blocker antigen, primary T cells (donor 1)transduced with HPV E7-TCR activator and ESO-LIR-1 blocker displaysimilar killing efficacy to primary T cells transduced with only the HPVE7-TCR activator. Luciferase measurement represents live target cells.RLU=relative light units; error bars indicate ±SD (n=2).

FIG. 24A is a pair of plots showing that A2-LIR-1 blocks Jurkatactivation in A2+ Raji cells but not WT Raji cells. Histograms show RajiWT “tumor” cells and Raji A2+“normal” cells have identical CD19 surfaceexpression while HLA-A*02 is expressed only in Raji A2 “normal” cells.

FIG. 24B is a plot showing that A2-LIR-1 blocks Jurkat activation in A2+Raji cells but not WT Raji cells. Jurkat cells transfected with eitherCD19 or CD19+A2-LIR-1 were co-cultured with either WT (A2-) Raji cellsor A2+ Raji cells at various cell ratios. RLU=relative light units,error bars indicate f SD (n=2).

FIGS. 25A-25B are each a series of images showing the reversibility ofblockade by LIR-1 inhibitory receptors. Primary T cells (donor 2)transduced with CD19 CAR activator and HLA-A*02 blocker demonstratereversible blockade (FIG. 25A) and activation (FIG. 25B) after 3 roundsof antigen exposure (AB-A-AB and A-AB-A) in in vitro cytotoxicity assayat 3:1 E:T. Primary T cell cytotoxicity assay was reproduced with threeHLA-A*02-negative donors. Images shown were captured at 72 hours.

FIGS. 25C-25D are each a pair of plots showing quantification of targetcell lysis (FIG. 25C) and IFNγ (FIG. 25D) in response to repeatedexposure to multiple rounds of normal and target cells, 3:1 E:T (T cellsfrom donor 2). Shown conditions are Untransduced, CD19-CAR T cells, andCD19-CAR T+A2-LIR-1. Error bars indicate ±SEM (n=2). *p<0.05, **p<0.01,***p<0.001, ****p<0.0001, determined using a two-way ANOVA followed byTukey's multiple-comparisons test. In this experiment, IFNγ responsediminished over time, while cytotoxicity remained robust.

FIGS. 26A-26B are each a pair of plots showing cytotoxic T cell killingand secretion of IFNγ in co-culture with a separate donor (donor 3).Cytotoxic CD19 CAR activator and HLA-A*02 blocker transduced T cellsdemonstrate reversible blocking after multiple rounds of antigenexposure in cytotoxic assays and IFNγ at 9:1 E:T. We noted that thisdonor's T cells survival and activity tailed off overtime in culture.Shown conditions are Untransduced, CD19-CAR T cells, and CD19-CART+A2-LIR-1. Cytotoxicity (FIG. 26A) and IFNγ (FIG. 26B) resultscorrespond to FIGS. 25C-25D. Error bars indicate SEM (n=2). *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001, determined using a two-way ANOVAfollowed by Tukey's multiple-comparisons test.

FIG. 27A is a plot showing primary T cells transduced with CD19 CARactivator and HLA-A*02 blocker demonstrates ˜20-fold expansion withCD3/28 stimulation over 10 days.

FIG. 27B is a diagram showing an experiment to show that CAR-T cellsexpressing a LIR-1 blocker receptor selectively kill tumors in axenograft model. HLA-A*02 NSG mice were administered either “tumorcells” (A2-negative Raji cells) or “normal cells” (A2-positive Rajicells) subcutaneously and primary T cells (human, HLA-A*02-negativedonor 4) were injected into the tail vein when Raji xenografts averaged˜70 mm³.

FIGS. 27C-27E are each a pair of plots that show readouts by calipermeasurement (FIG. 27C), human T cell counts in peripheral blood by flowcytometry (FIG. 27D), and survival (FIG. 27E). Error bars in C-Dindicate f SEM (n=7). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001,determined using a two-way ANOVA followed by Tukey'smultiple-comparisons test.

FIG. 28A is a series of plots showing flow cytometry analysis of primaryT cell post-enrichment and expansion prepared for mouse tail veininjection. Tumor volume was measured at the number of days from T cellinjection, starting 10 days prior to T cell injection (−10) to 40 daysfollowing T cell injection (40). Tumor volumes ranged from 0 to 2500mm³.

FIG. 28B is a series of plots showing tumor measurement by caliperplotted for individual mice in each group.

FIG. 28C is a series of plots showing correlation of huCD3+ T cells inmouse blood to tumor growth. Graph of huCD3+ T cells compared to tumorvolume 10 days and 17 days after T cell injection.

FIG. 28D is a pair of plots showing huCD4+ and huCD8+ T cell counts inperipheral blood by flow cytometry. Open circles, mice grafted with“normal” cells; closed circle, mice grafted with tumor cells. Sampleswith fewer than 100 cells were excluded from the analysis. Error barsindicate f SEM (n=7 in all groups except n=6 in CD19+/A2− Raji grouptreated with CD19-CAR+A2-LIR-1 T cells).

FIG. 29A is a series of images showing histological analysis of T cellinfiltration in tumors. Representative images of tumor samples collectedat study termination, sectioned and stained for huCD3 are shown.

FIG. 29B is a plot showing quantification of T cell infiltration usingImageJ. T cell infiltration was significantly higher for T cells withCD19-CAR or CD19-CAR+A2-LIR-1 in CD19+/A2− tumors compared tountransduced cells. However, in CD19+/A2+ tumors, CD19-CAR+A2-LIR-1 Tcells were not significantly different compared to untransduced cells.There was also a significant drop in infiltration of CD19-CAR+A2-LIR-1 Tcells between CD19+/A2− and CD19+/A2+ tumors. Qualitatively,CD19-CAR+A2-LIR-1 T cells were less prevalent in CD19+/A2+ tumorscompared to CD19-CAR only T cells; however, this difference was notstatistically significant. Saline samples were similarly quantified toshow background staining levels. Groups of data were analyzed using anordinary one-way ANOVA, while individual pairs between “tumor” and“normal” were analyzed using an unpaired t test. ns=not significant,*p<0.05, **p<0.01.

DETAILED DESCRIPTION

The present disclosure describes receptors having one or more domainsfrom Leukocyte immunoglobulin-like receptor subfamily B member 1(LILRB1, sometimes referred to as LIR1 or LIR-1). Numerous receptors,engineered cells, and uses thereof are contemplated herein. Theinventors have found that chimeric receptors comprising anantigen-binding domain and one or more LILRB1 domains, including theLILRB1 intracellular domain, can inhibit immune cell signaling even inthe presence of activatory chimeric antigen receptors (CARs) or T cellreceptors (TCRs).

The term “chimeric antigen receptors” or “CARs” as used herein, mayrefer to artificial T-cell receptors, chimeric T-cell receptors, orchimeric immunoreceptors, for example, and encompass engineeredreceptors that graft an artificial specificity onto a particular immuneeffector cell, such as a helper T cell (CD4+), cytotoxic T cell (CD8+)or NK cell. CARs may be employed to impart the specificity of amonoclonal antibody onto a T cell, thereby allowing a large number ofspecific T cells to be generated, for example, for use in adoptive celltherapy. In specific embodiments, CARs direct specificity of the cell toa tumor associated antigen. In some embodiments, CARs comprise anintracellular signaling domain, a transmembrane domain, and anextracellular domain comprising an antigen-binding region. In someembodiments, CARs comprise fusions of single-chain variable fragments(scFvs) or scFabs derived from monoclonal antibodies, fused to atransmembrane domain and intracellular signaling domain(s). The fusionmay also comprise a hinge. Either heavy-light (H-L) and light-heavy(L-H) scFvs may be used. The specificity of CAR designs may be derivedfrom ligands of receptors (e.g., peptides). Depending on the type ofintracellular domain, a CAR can be an activatory receptor or aninhibitory receptor. In some embodiments, for example when the CAR is anactivatory receptor, the CAR comprises domains for additionalco-stimulatory signaling, such as CD3, FcR, CD27, CD28, CD137, DAP10,and/or 0X40. In some embodiments, molecules can be co-expressed with theCAR, including co-stimulatory molecules, reporter genes for imaging(e.g., for positron emission tomography), gene products thatconditionally ablate the T cells upon addition of a prodrug, homingreceptors, cytokines, and cytokine receptors. As used herein,characteristics attributed to a chimeric antigen receptor may beunderstood to refer to the receptor itself or to a host cell comprisingthe receptor.

As used herein, a “TCR”, sometimes also called a “TCR complex” or“TCR/CD3 complex” refers to a protein complex comprising a TCR alphachain, a TCR beta chain, and one or more of the invariant CD3 chains(zeta, gamma, delta and epsilon), sometimes referred to as subunits. TheTCR alpha and beta chains can be disulfide-linked to function as aheterodimer to bind to peptide-MHC complexes. Once the TCR alpha/betaheterodimer engages peptide-MHC, conformational changes in the TCRcomplex in the associated invariant CD3 subunits are induced, whichleads to their phosphorylation and association with downstream proteins,thereby transducing a primary stimulatory signal. In an exemplary TCRcomplex, the TCR alpha and TCR beta polypeptides form a heterodimer, CD3epsilon and CD3 delta form a heterodimer, CD3 epsilon and CD3 gamma fora heterodimer, and two CD3 zeta form a homodimer.

The term “stimulation” refers to a primary response induced by bindingof a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3complex) with its cognate ligand thereby mediating a signal transductionevent, such as, but not limited to, signal transduction via the TCR/CD3complex. Stimulation can mediate altered expression of certainmolecules, and/or reorganization of cytoskeletal structures, and thelike.

The term “stimulatory molecule” or “stimulatory domain” refers to amolecule or portion thereof that, when natively expressed by a T-cell,provides the primary cytoplasmic signaling sequence(s) that regulateactivation of the TCR complex in a stimulatory way for at least someaspect of the T-cell signaling pathway. TCR alpha and/or TCR beta chainsof wild type TCR complexes do not contain stimulatory domains andrequire association with CD3 subunits such as CD3 zeta to initiatesignaling. In one aspect, the primary stimulatory signal is initiatedby, for instance, binding of a TCR/CD3 complex with an a majorhistocompatibility complex (MHC) bound to peptide, and which leads tomediation of a T-cell response, including, but not limited to,proliferation, activation, differentiation, and the like. One or morestimulatory domains, as described herein, can be fused to theintracellular portion of any one or more subunits of the TCR complex,including TCR alpha, TCR beta, CD3 delta, CD3 gamma and CD3 epsilon.

As used herein, a “domain capable of providing a stimulatory signal”refers to any domain that, either directly or indirectly, can provide astimulatory signal that enhances or increases the effectiveness ofsignaling mediated by the TCR complex to enhance at least some aspect ofT-cell signaling. The domain capable of providing a stimulatory signalcan provide this signal directly, for example with the domain capable ofproviding the stimulatory signal is a primary stimulatory domain orco-stimulatory domain. Alternatively, or in addition, the domain capableof providing the stimulatory signal can act indirectly. For example, thedomain can be a scaffold that recruits stimulatory proteins to the TCR,or provide an enzymatic activity, such as kinase activity, that actsthrough downstream targets to provide a stimulatory signal.

As used herein, a “domain capable of providing an inhibitory signal”refers to any domain that, either directly or indirectly, can provide aninhibitory signal that inhibits or decreases the effectiveness signalingmediated by the TCR complex. The domain capable of providing aninhibitory signal can reduce, or block, totally or partially, at leastsome aspect of T-cell signaling or function. The domain capable ofproviding an inhibitory signal can provide this signal directly, forexample with the domain capable of providing the inhibitory signalprovides a primary inhibitory signal. Alternatively, or in addition, thedomain capable of providing the stimulatory signal can act indirectly.For example, the domain can recruit additional inhibitory proteins tothe TCR, or can provide an enzymatic activity that acts throughdownstream targets to provide an inhibitory signal.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Asanother example, a range such as 95-99% identity, includes somethingwith 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This appliesregardless of the breadth of the range.

In general, “sequence identity” or “sequence homology” refers to anexact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Typically, techniques for determining sequence identityinclude determining the nucleotide sequence of a polynucleotide and/ordetermining the amino acid sequence encoded thereby and comparing thesesequences to a second nucleotide or amino acid sequence. Two or moresequences (polynucleotide or amino acid) can be compared by determiningtheir “percent identity.” The percent identity of two sequences, whethernucleic acid or amino acid sequences, is the number of exact matchesbetween two aligned sequences divided by the length of the shortersequences and multiplied by 100. Percent identity may also bedetermined, for example, by comparing sequence information using theadvanced BLAST computer program, including version 2.2.9, available fromthe National Institutes of Health. The BLAST program is based on thealignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol.215:403-410 (1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res.25:3389-3402 (1997). Briefly, the BLAST program defines identity as thenumber of identical aligned symbols (generally nucleotides or aminoacids), divided by the total number of symbols in the shorter of the twosequences. The program may be used to determine percent identity overthe entire length of the proteins being compared. Default parameters areprovided to optimize searches with short query sequences in, forexample, with the blastp program. Ranges of desired degrees of sequenceidentity are approximately 80% to 100% and integer values therebetween.Typically, the percent identities between a disclosed sequence and aclaimed sequence are at least 80%, at least 85%, at least 90%, at least95%, or at least 98%.

As used herein, a “subsequence” refers to a length of contiguous aminoacids or nucleotides that form a part of a sequence described herein. Asubsequence may be identical to a part of a full length sequence whenaligned to the full length sequence, or less than 100% identical to thepart of the full length sequence to which it aligns (e.g., 90% identicalto 50% of the full sequence, or the like).

The term “exogenous” is used herein to refer to any molecule, includingnucleic acids, protein or peptides, small molecular compounds, and thelike that originate from outside the organism. In contrast, the term“endogenous” refers to any molecule that originates from inside theorganism (i.e., naturally produced by the organism).

A polynucleotide is “operably linked” to another polynucleotide when itis placed into a functional relationship with the other polynucleotide.For example, a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence. A peptide is“operably linked” to another peptide when the polynucleotides encodingthem are operably linked, preferably they are in the same open readingframe.

A “promoter” is a sequence of DNA needed to turn a gene on or off.Promoters are located immediately upstream and/or overlapping thetranscription start site, and are usually between about one hundred toseveral hundred base pairs in length.

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control. However, mention of any reference,article, publication, patent, patent publication, and patent applicationcited herein is not, and should not be taken as an acknowledgment, orany form of suggestion, that they constitute valid prior art or formpart of the common general knowledge in any country in the world.

In the present description, any concentration range, percentage range,ratio range, or integer range is to be understood to include the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated. The term “about”, when immediately preceding anumber or numeral, means that the number or numeral ranges plus or minus10%.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

Leukocyte Immunoglobulin-Like Receptor Subfamily B Member 1 (LILRB1)

The present disclosure describes receptors having one or more domainsfrom Leukocyte immunoglobulin-like receptor subfamily B member 1(LILRB1, or LIR1). Numerous receptors, engineered cells, and usesthereof are contemplated herein.

Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1),also known as Leukocyte immunoglobulin-like receptor B1, as well asILT2, LIR1, MIR7, PIRB, CD85J, ILT-2 LIR-1, MIR-7 and PIR-B, is a memberof the leukocyte immunoglobulin-like receptor (LIR) family. The LILRB1protein belongs to the subfamily B class of LIR receptors. Thesereceptors contain two to four extracellular immunoglobulin domains, atransmembrane domain, and two to four cytoplasmic immunoreceptortyrosine-based inhibitory motifs (ITIMs). The LILRB1 receptor isexpressed on immune cells, where it binds to MHC class I molecules onantigen-presenting cells and transduces a negative signal that inhibitsstimulation of an immune response. LILRB1 is thought to regulateinflammatory responses, as well as cytotoxicity, and to play a role inlimiting auto-reactivity. Multiple transcript variants encodingdifferent isoforms of LILRB1 exist, all of which are contemplated aswithin the scope of the instant disclosure.

In some embodiments of the receptors having one or domains of LILRB1,the one or more domains of LILRB1 comprise an amino acid sequence thatis at least 80%/o, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or is identical to a sequence orsubsequence of SEQ ID NO: 1. In some embodiments, the one or moredomains of LILRB1 comprise an amino acid sequence that is identical to asequence or subsequence of SEQ ID NO: 1. In some embodiments, the one ormore domains of LILRB1 consist of an amino acid sequence that is atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or is identical to a sequence or subsequence ofSEQ ID NO: 1. In some embodiments, the one or more domains of LILRB1consist of an amino acid sequence that is identical to a sequence orsubsequence of SEQ ID NO: 1.

In some embodiments of the receptors having one or domains of LILRB1,the one or more domains of LILRB1 are encoded by a polynucleotidesequence that is at least 80%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or is identical to a sequenceor subsequence of SEQ ID NO: 34.

In some embodiments of the receptors having one or domains of LILRB1,the one or more domains of LILRB1 are encoded by a polynucleotidesequence that is identical to a sequence or subsequence of SEQ ID NO:34.

Receptors

In various embodiments, a chimeric antigen receptor is provided,comprising a polypeptide, wherein the polypeptide comprises one or moreof: an LILRB1 hinge domain or functional fragment or variant thereof; anLILRB1 transmembrane domain or a functional variant thereof; and anLILRB1 intracellular domain or an intracellular domain comprising atleast one, or at least two immunoreceptor tyrosine-based inhibitorymotifs (ITIMs), wherein each ITIM is independently selected from NLYAAV(SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), andSIYATL (SEQ ID NO: 11).

Intracellular Domain

The disclosure provides chimeric antigen receptors, the chimeric antigenreceptors comprising a polypeptide. In some embodiments, the polypeptidecomprises an intracellular domain. In some embodiments, theintracellular domain is an LILRB1 intracellular domain or a functionalvariant thereof.

As used herein, “intracellular domain” refers to the cytoplasmic orintracellular domain of a protein, such as a receptor, that interactswith the interior of the cell, and carries out a cytosolic function. Asused herein, “cytosolic function” refers to a function of a protein orprotein complex that is carried out in the cytosol of a cell. Forexample, intracellular signal transduction cascades are cytosolicfunctions.

As used herein an “immunoreceptor tyrosine-based inhibitory motif” or“ITIM” refers to a conserved sequence of amino acids with a consensussequence of S/I/V/LxYxxI/V/L (SEQ ID NO. 124), or the like, that isfound in the cytoplasmic tails of many inhibitory receptors of theimmune system. After ITIM-possessing inhibitory receptors interact withtheir ligand, the ITIM motif is phosphorylated, allowing the inhibitoryreceptor to recruit other enzymes, such as the phosphotyrosinephosphatases SHP-1 and SHP-2, or the inositol-phosphatase called SHIP.

In some embodiments, the polypeptide comprises an intracellular domaincomprising at least one immunoreceptor tyrosine-based inhibitory motif(ITIM), at least two ITIMs, at least 3 ITIMs, at least 4 ITIMs, at least5 ITIMs or at least 6 ITIMs. In some embodiments, the intracellulardomain has 1, 2, 3, 4, 5, or 6 ITIMs.

In some embodiments, the polypeptide comprises an intracellular domaincomprising at least one ITIM selected from the group of ITIMs consistingof NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10),and SIYATL (SEQ ID NO: 11).

In further particular embodiments, the polypeptide comprises anintracellular domain comprising at least two immunoreceptortyrosine-based inhibitory motifs (ITIMs), wherein each ITIM isindependently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

In some embodiments, the intracellular domain comprises both ITIMsNLYAAV (SEQ ID NO: 8) and VTYAEV (SEQ ID NO: 9). In some embodiments,the intracellular domain comprises a sequence at least 95% identical toSEQ ID NO: 12. In some embodiments, the intracellular domain comprisesor consists essentially of a sequence identical to SEQ ID NO: 12.

In some embodiments, the intracellular domain comprises both ITIMsVTYAEV (SEQ ID NO: 9) and VTYAQL (SEQ ID NO: 10). In some embodiments,the intracellular domain comprises a sequence at least 95% identical toSEQ ID NO: 13. In some embodiments, the intracellular domain comprisesor consists essentially of a sequence identical to SEQ ID NO: 13.

In some embodiments, the intracellular domain comprises both ITIMsVTYAQL (SEQ ID NO: 10) and SIYATL (SEQ ID NO: 11). In some embodiments,the intracellular domain comprises a sequence at least 95% identical toSEQ ID NO: 14. In some embodiments, the intracellular domain comprisesor consists essentially of a sequence identical to SEQ ID NO: 14.

In some embodiments, the intracellular domain comprises the ITIMs NLYAAV(SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), and VTYAQL (SEQ ID NO: 10). Insome embodiments, the intracellular domain comprises a sequence at least95% identical to SEQ ID NO: 15. In some embodiments, the intracellulardomain comprises or consists essentially of a sequence identical to SEQID NO: 15.

In some embodiments, the intracellular domain comprises the ITIMs VTYAEV(SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). Insome embodiments, the intracellular domain comprises a sequence at least95% identical to SEQ ID NO: 16. In some embodiments, the intracellulardomain comprises or consists essentially of a sequence identical to SEQID NO: 16.

In some embodiments, the intracellular domain comprises the ITIMs NLYAAV(SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), andSIYATL (SEQ ID NO: 11). In embodiments, the intracellular domaincomprises a sequence at least 95% identical to SEQ ID NO: 17. In someembodiments, the intracellular domain comprises or consists essentiallyof a sequence identical to SEQ ID NO: 17.

In some embodiments, the intracellular domain comprises a sequence atleast 95% identical to the LILRB1 intracellular domain (SEQ ID NO: 7).In some embodiments, the intracellular domain comprises or consistsessentially of a sequence identical to the LILRB1 intracellular domain(SEQ ID NO: 7).

LILRB1 intracellular domains or functional variants thereof of thedisclosure can have at least 1, at least 2, at least 4, at least 4, atleast 5, at least 6, at least 7, or at least 8 ITIMs. In someembodiments, the LILRB1 intracellular domain or functional variantthereof has 2, 3, 4, 5, or 6 ITIMs.

In particular embodiments, the polypeptide comprises an intracellulardomain comprising two, three, four, five, or six immunoreceptortyrosine-based inhibitory motifs (ITIMs), wherein each ITIM isindependently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

In particular embodiments, the polypeptide comprises an intracellulardomain comprising at least three immunoreceptor tyrosine-basedinhibitory motifs (ITIMs), wherein each ITIM is independently selectedfrom NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO: 11).

In particular embodiments, the polypeptide comprises an intracellulardomain comprising three immunoreceptor tyrosine-based inhibitory motifs(ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ IDNO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQID NO: 11).

In particular embodiments, the polypeptide comprises an intracellulardomain comprising four immunoreceptor tyrosine-based inhibitory motifs(ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ IDNO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQID NO: 11).

In particular embodiments, the polypeptide comprises an intracellulardomain comprising five immunoreceptor tyrosine-based inhibitory motifs(ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ IDNO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQID NO: 11).

In particular embodiments, the polypeptide comprises an intracellulardomain comprising six immunoreceptor tyrosine-based inhibitory motifs(ITIMs), wherein each ITIM is independently selected from NLYAAV (SEQ IDNO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQID NO: 11).

In particular embodiments, the polypeptide comprises an intracellulardomain comprising at least seven immunoreceptor tyrosine-basedinhibitory motifs (ITIMs), wherein each ITIM is independently selectedfrom NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL (SEQ ID NO:10), and SIYATL (SEQ ID NO: 11).

In some embodiments, the intracellular domain comprises a TCR alphaintracellular domain. In some embodiments, the intracellular domaincomprises a TCR alpha intracellular domain and an LILRB1 intracellulardomain, as described herein. In some embodiments, a TCR alphaintracellular domain comprises Ser-Ser. In some embodiments, a TCR alphaintracellular domain is encoded by a sequence of TCCAGC.

In some embodiments, the intracellular domain comprises a TCR betaintracellular domain. In some embodiments, the intracellular domaincomprises a TCR beta intracellular domain and an LILRB1 intracellulardomain, as described herein. In some embodiments, the TCR betaintracellular domain comprises an amino acid sequence having at least80% identity, at least 90% identity, or is identical to a sequence of:MAMVKRKDSR (SEQ ID NO: 94). In some embodiments, the TCR betaintracellular domain comprises, or consists essentially of MAMVKRKDSR(SEQ ID NO: 94). In some embodiments, the TCR beta intracellular domainis encoded by a sequence of ATGGCCATGGTCAAGAGAAAGGATTCCAGA (SEQ ID NO:95).

Transmembrane Domain

The disclosure provides chimeric antigen receptors the receptorscomprising a polypeptide. In some embodiments, the polypeptide comprisesa transmembrane domain. In some embodiments, the transmembrane domain isa LILRB1 transmembrane domain or a functional variant thereof.

A “transmembrane domain”, as used herein, refers to a domain of aprotein that spans membrane of the cell. Transmembrane domains typicallyconsist predominantly of non-polar amino acids, and may traverse thelipid bilayer once or several times. Transmembrane domains usuallycomprise alpha helices, a configuration which maximizes internalhydrogen bonding.

Transmembrane domains isolated or derived from any source are envisagedas within the scope of the fusion proteins of the disclosure.

In particular embodiments, the polypeptide comprises an LILRB1transmembrane domain or a functional variant thereof.

In some embodiments, the LILRB1 transmembrane domain or a functionalvariant thereof comprises a sequence at least 95% identical, at least96% identical, at least 97% identical, at least 98% identical or atleast 99% to SEQ ID NO: 5. In some embodiments, the LILRB1 transmembranedomain or a functional variant thereof comprises a sequence at least 95%identical to SEQ ID NO: 5. In some embodiments, the LILRB1 transmembranedomain comprises a sequence identical to SEQ ID NO: 5. In embodiments,the LILRB1 transmembrane domain consists essentially of a sequenceidentical to SEQ ID NO: 5.

In some embodiments of the chimeric antigen receptors of the disclosure,the transmembrane domain is not a LILRB1 transmembrane domain. In someembodiments, the transmembrane domain is one that is associated with oneof the other domains of the fusion protein, or isolated or derived fromthe same protein as one of the other domains of the fusion protein.

The transmembrane domain may be derived either from a natural or from arecombinant source. Where the source is natural, the domain may bederived from any membrane-bound or transmembrane protein. Exemplarytransmembrane domains may include at least the transmembrane region(s)of e.g., the alpha, beta or zeta chain of the TCR, CD3 delta, CD3epsilon or CD3 gamma, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16,CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

In some embodiments, the transmembrane comprises a TCR alphatransmembrane domain. In some embodiments, the TCR alpha transmembranedomain comprises an amino acid sequence having at least 85% identity, atleast 90% identity, at least 95% identity, at least 96% identity, atleast 97% identity, at least 98% identity, at least 99% identity or isidentical to a sequence of: VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 96). Insome embodiments, the TCR alpha transmembrane domain comprises, orconsists essentially of, VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 96). Insome embodiments, the TCR alpha transmembrane domain is encoded by asequence of:

(SEQ ID NO: 97) GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGG.

In some embodiments, the transmembrane comprises a TCR betatransmembrane domain. In some embodiments, the TCR beta transmembranedomain comprises an amino acid sequence having at least 85% identity, atleast 90% identity, at least 95% identity, at least 96% identity, atleast 97% identity, at least 98% identity, at least 99% identity or isidentical to a sequence of: TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 98). Insome embodiments, the TCR beta transmembrane domain comprises, orconsists essentially of TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 98). In someembodiments, the TCR beta transmembrane domain is encoded by a sequenceof

(SEQ ID NO: 99) ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTG.

In some embodiments, the TCR alpha and/or TCR beta transmembrane domaincomprises one or more mutations that attenuate or abolish interaction ofthe TCR with the TCR CD3 subunit. In some embodiments, the TCR alphatransmembrane domain comprises a R253L mutation. In some embodiments,the TCR beta transmembrane domain comprises a K288L mutation.

In some embodiments the transmembrane domain comprise a CD28transmembrane domain. In some embodiments, the CD28 transmembrane domaincomprises an amino acid sequence having at least 80% identity, at least90% identity, at least 95% identity, at least 99% identity or isidentical to a sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 100).In some embodiments, the CD28 transmembrane domain comprises or consistsessentially of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 100). In someembodiments, the CD28 transmembrane domain is encoded by a nucleotidesequence having at least 80% identity, at least 90% identity, at least95% identity, at least 99% identity or is identical to a sequence of

(SEQ ID NO: 101) TTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACAGTGGCCTTCATCATCTTTT GGGTG.

In some embodiments, the transmembrane domain can be attached to theextracellular region chimeric antigen receptor, e.g., theantigen-binding domain or ligand binding domain, via a hinge, e.g., ahinge from a human protein. For example, in some embodiments, the hingecan be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, a CD8ahinge or an LILRB1 hinge.

Hinge Domain

The disclosure provides chimeric antigen receptors, the receptorscomprising a polypeptide. In some embodiments, the polypeptide comprisesa hinge domain. In some embodiments, the hinge domain is a LILRB1 hingedomain or a functional variant thereof.

The LILRB1 protein has four immunoglobulin (Ig) like domains termed D1,D2, D3 and D4. In some embodiments, the LILRB1 hinge domain comprises anLILRB1 D3D4 domain or a functional variant thereof. In some embodiments,the LILRB1 D3D4 domain comprises a sequence at least 95%, at least 96%,at least 97%, at least 98%, at least 99% or identical to SEQ ID NO: 18.In some embodiments, the LILRB1 D3D4 domain comprises or consistsessentially of SEQ ID NO: 18.

In some embodiments, the polypeptide comprises the LILRB1 hinge domainor functional fragment or variant thereof. In embodiments, the LILRB1hinge domain or functional fragment or variant thereof comprises asequence at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identical or identical to SEQ ID NO: 4, SEQ ID NO: 18, or SEQID NO: 19. In embodiments, the LILRB1 hinge domain or functionalfragment or variant thereof comprises a sequence at least 95% identicalto SEQ ID NO: 4, SEQ ID NO: 18, or SEQ ID NO: 19.

In some embodiments, the LILRB1 hinge domain comprises a sequenceidentical to SEQ ID NO: 4, SEQ ID NO: 18, or SEQ ID NO: 19.

In some embodiments, the LILRB1 hinge domain consists essentially of asequence identical to SEQ ID NO: 4, SEQ ID NO: 18, or SEQ ID NO: 19.

In some embodiments the chimeric antigen receptors of the disclosure,the polypeptide comprises a hinge that is not isolated or derived fromLILRB1.

In some embodiments, the hinge is isolated or derived from CD8a or CD28.In some embodiments, the CD8a hinge comprises an amino acid sequencehaving at least 80% identity, at least 90% identity, at least 95%identity, at least 99% identity or is identical to a sequence ofTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 102). In someembodiments, the CD8a hinge comprisesTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 102). In someembodiments, the CD8a hinge consists essentially ofTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 102). In someembodiments, the CD8a hinge is encoded by a nucleotide sequence havingat least 80% identity, at least 90% identity, at least 95% identity, atleast 99% identity or is identical to a sequence

{SEQ ID NO: 103) accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggac ttcgcctgtgat.

In some embodiments, the CD28 hinge comprises an amino acid sequencehaving at least 80% identity, at least 90% identity, at least 95%identity, at least 99% identity or is identical to a sequence ofCTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 104). In someembodiments, the CD28 hinge comprises or consists essentially ofCTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 104). In someembodiments, the CD28 hinge is encoded by a nucleotide sequence havingat least 80% identity, at least 90% identity, at least 95% identity, atleast 99% identity or is identical to a sequence of

(SEQ ID NO: 105) tgtaccattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctattteccggaccttctaagccc.

Combinations of LILRB1 Domains

In some embodiments, the chimeric antigen receptors of the disclosurecomprise a polypeptide comprising more than one LILRB1 domain orfunctional equivalent thereof. For example, in some embodiments, thepolypeptide comprises an LILRB1 transmembrane domain and intracellulardomain, or an LILRB1 hinge domain, transmembrane domain andintracellular domain.

In particular embodiments, the polypeptide comprises an LILRB1 hingedomain or functional fragment or variant thereof, and the LILRB1transmembrane domain or a functional variant thereof. In someembodiments, the polypeptide comprises a sequence at least 95%identical, at least 96% identical, at least 97% identical, at least 98%identical, at least 99% identical or identical to SEQ ID NO: 20. In someembodiments, the polypeptide comprises a sequence at least 95% identicalto SEQ ID NO: 20. In some embodiments, the polypeptide comprises asequence identical to SEQ ID NO: 20.

In further embodiments, the polypeptide comprises: the LILRB1transmembrane domain or a functional variant thereof, and an LILRB1intracellular domain and/or an intracellular domain comprising at leastone immunoreceptor tyrosine-based inhibitory motif (ITIM), wherein theITIM is selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9),VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11). In some embodiments,the polypeptide comprises the LILRB1 transmembrane domain or afunctional variant thereof, and an LILRB1 intracellular domain and/or anintracellular domain comprising at least two ITIM, wherein each ITIM isindependently selected from NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO:9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

In some embodiments, the polypeptide comprises a LILRB1 transmembranedomain and intracellular domain. In some embodiments, the polypeptidecomprises a sequence at least 95% identical, at least 96% identical, atleast 97% identical, at least 98% identical, at least 99% identical oridentical to SEQ ID NO: 21. In some embodiments, the polypeptidecomprises a sequence at least 95% identical to SEQ ID NO: 21. In someembodiments, the polypeptide comprises a sequence identical to SEQ IDNO: 21.

In preferred embodiments, the polypeptide comprises: an LILRB1 hingedomain or functional fragment or variant thereof; an LILRB1transmembrane domain or a functional variant thereof; and an LILRB1intracellular domain and/or an intracellular domain comprising at leasttwo immunoreceptor tyrosine-based inhibitory motifs (ITIMs), whereineach ITIM is independently selected from LYAAV (SEQ ID NO: 8), VTYAE(SEQ ID NO:9), VTYAQL (SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).

In some embodiments, the polypeptide comprises a sequence at least 95%identical to SEQ ID NO: 2 or SEQ ID NO: 3, or at least 99% identical toSEQ ID NO: 2 or SEQ ID NO: 3, or identical to SEQ ID NO: 2 or SEQ ID NO:3.

In some embodiments, the polypeptide comprises a sequence at least 99%identical to SEQ ID NO. 20, or at least 99% identical to SEQ ID NO: 20,or identical to SEQ ID NO: 20.

In some embodiments, the polypeptide comprises a sequence at least 99%identical to SEQ ID NO: 21, or at least 99% identical to SEQ ID NO: 21,or identical to SEQ ID NO: 21.

Extracellular Domains

The disclosure provides chimeric antigen receptors comprising apolypeptide. In some embodiments, the polypeptide comprises a ligandbinding domain, such as an antigen-binding domain. Suitableantigen-binding domains include, but are not limited to antigen-bindingdomains from antibodies, antibody fragments, scFv, antigen-bindingdomains derived from T cell receptors, and the like. All forms ofantigen-binding domains known in the art are envisaged as within thescope of the disclosure.

An “extracellular domain”, as used herein, refers to the extracellularportion of a protein. For example, the TCR alpha and beta chains eachcomprise an extracellular domain, which comprise a constant and avariable region involved in peptide-MHC recognition. The “extracellulardomain” can also comprise a fusion domain, for example of fusionsbetween additional domains capable of binding to and targeting aspecific antigen and the endogenous extracellular domain of the TCRsubunit.

The term “antibody,” as used herein, refers to a protein, or polypeptidesequences derived from an immunoglobulin molecule, which specificallybinds to an antigen. Antibodies can be intact immunoglobulins ofpolyclonal or monoclonal origin, or fragments thereof and can be derivedfrom natural or from recombinant sources.

The terms “antibody fragment” or “antibody binding domain” refer to atleast one portion of an antibody, or recombinant variants thereof, thatcontains the antigen-binding domain, i.e., an antigenic determiningvariable region of an intact antibody, that is sufficient to conferrecognition and specific binding of the antibody fragment to a target,such as an antigen and its defined epitope. Examples of antibodyfragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fvfragments, single-chain (sc)Fv (“scFv”) antibody fragments, linearantibodies, single domain antibodies (abbreviated “sdAb”) (either VL orVH), camelid VHH domains, and multi-specific antibodies formed fromantibody fragments.

The term “scFv” refers to a fusion protein comprising at least oneantibody fragment comprising a variable region of a light chain and atleast one antibody fragment comprising a variable region of a heavychain, wherein the light and heavy chain variable regions arecontiguously linked via a short flexible polypeptide linker, and capableof being expressed as a single polypeptide chain, and wherein the scFvretains the specificity of the intact antibody from which it is derived.

“Heavy chain variable region” or “VH” (or, in the case of single domainantibodies, e.g., nanobodies, “VHH”) with regard to an antibody refersto the fragment of the heavy chain that contains three CDRs interposedbetween flanking stretches known as framework regions, these frameworkregions are generally more highly conserved than the CDRs and form ascaffold to support the CDRs.

Unless specified, as used herein a scFv may have the VL and VH variableregions in either order, e.g., with respect to the N-terminal andC-terminal ends of the polypeptide, the scFv may comprise VL-linker-VHor may comprise VH-linker-VL.

The term “antibody light chain,” refers to the smaller of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations. Kappa (“K”) and lambda (“λ”) light chains referto the two major antibody light chain isotypes.

The term “recombinant antibody” refers to an antibody that is generatedusing recombinant DNA technology, such as, for example, an antibodyexpressed by a bacteriophage or yeast expression system. The term shouldalso be construed to mean an antibody which has been generated by thesynthesis of a DNA molecule encoding the antibody and which DNA moleculeexpresses an antibody protein, or an amino acid sequence specifying theantibody, wherein the DNA or amino acid sequence has been obtained usingrecombinant DNA or amino acid sequence technology which is available andwell known in the art.

In some embodiments, for example those embodiments wherein the receptorcomprises a first and a second polypeptide, the antigen-binding domainis isolated or derived from a T cell receptor (TCR) extracellular domainor an antibody.

In preferred embodiments, the polypeptide comprises antigen-bindingdomain, e.g., an antigen-binding domain other than the LILRB1antigen-binding protein. An illustrative embodiments of receptor havinga single antigen-binding domain is depicted in FIG. 1. The disclosurecontemplated chimeric antigen receptors have two, three, four or moreantigen-binding domains. The antigen-binding domains may be provided onthe same or a different chain of the chimeric antigen receptor. Inembodiments, the chimeric antigen receptor is a DARIC as described, forexample in Leung et al. JCI Insight. 2019 Jun. 6; 4(11):e124430,WO2015017214A1; and WO2017156484A1.

In some embodiments, the receptor is an inhibitory chimeric antigenreceptor (iCAR). Various methods and composition suitable for use withthe embodiments disclosure herein include those provided inUS2018/0044399A1; WO2018148454A1; and WO2017087723A1, each of which isincorporated herein for all purposes.

In some embodiments, the antigen-binding domain comprises a single chainvariable fragment (scFv).

In some embodiments, the receptor comprises a second polypeptide. Thedisclosure provides receptors having two polypeptides each having a partof a ligand-binding domain (e.g. cognates of a heterodimeric LDB, suchas a TCRα/β- or Fab-based LBD) and each having an intracellular domain,as depicted in FIG. 2A. The disclosure further provides receptors havingtwo polypeptides, each having a part of a ligand-binding domain (e.g.cognates of a heterodimeric LDB, such as a TCRα/β- or Fab-based LBD) andone part of the ligand binding domain is fused to a hinge ortransmembrane domain, while the other part of the ligand binding domainhas no intracellular domain, as depicted in FIG. 2B. Further variationsinclude receptors where each polypeptide has a hinge domain, and whereeach polypeptide has a hinge and transmembrane domain. In someembodiments, the hinge domain is absent. In other embodiments, the hingedomain is a membrane proximal extracellular region (MPER), such as theLILRB1 D3D4 domain. In any of the embodiments disclosed herein, thedomains may be fused adjacent to one another with linkers between them.

In some embodiments, the first polypeptide comprises a first chain of anantibody and the second polypeptide comprise a second chain of saidantibody.

In some embodiments, the receptor comprises a Fab fragment of anantibody. In embodiments, an antigen-binding fragment of the heavy chainof the antibody, and the second polypeptide comprises an antigen-bindingfragment of the light chain of the antibody. In embodiments, the firstpolypeptide comprises an antigen-binding fragment of the light chain ofthe antibody, and the second polypeptide comprises an antigen-bindingfragment of the heavy chain of the antibody.

In some embodiments, the first polypeptide comprises a first chain of aT-cell receptor (TCR) and the second polypeptide comprises a secondchain of said TCR. In embodiments, the receptor comprises anextracellular fragment of a T cell receptor (TCR). In embodiments, thefirst polypeptide comprises an antigen-binding fragment of the alphachain of the TCR, and the second polypeptide comprises anantigen-binding fragment of the beta chain of the TCR. In someembodiments, the first polypeptide comprises an antigen-binding fragmentof the beta chain of the TCR, and the second polypeptide comprises anantigen-binding fragment of the alpha chain of the TCR.

In some embodiments, the receptor comprises a single-chain TCR, such as,without limitation, those disclosed in WO2017091905A1.

Illustrative Antigen-Binding Domains

Various single variable domains known in the art or disclosed herein aresuitable for use in embodiments. Such scFv's include, for example andwithout limitation the following mouse and humanized scFv antibodiesthat bind HLA-A*02 in a peptide-independent way (complementaritydetermining regions underlined):

C-001765 MMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDPTLKISRVEAEPLGVYYCFQGSHVPRTSGGGTKLEIKGGGGSGGGGSGGGGSGGQVQLQQSGPELVK PGASVRISCKASGYTFTSYHIHWVKQRPGQGLEWIGWIYPGNVNTEYNEKFKGKATLTAPKSSSTAYMHL SSLTSEPSAVYFCAREEITYAMDYWGQGTSVTVSSYG; (SEQ ID NO: 35) or PVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGS GSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTSGGGTKLEIKGGGGSGGGGSGGGGSGGQVQLQQSGPEL VKPGASVRISCKASGYTFTSYHIHWVKQRPGQGLEWIGWIYPGNVNTEYNEKFKGKATLTADKSSSTAYM HLSSLTSEDSAVYFCAREEITYAMDYWGQGTSVTVSS (SEQ ID NO. 125, the correspondingPolynucleotide sequence is provided as SEQ ID NO: 127) C-002159QLVQSGAEVKKPGSSVKVSCKASGYTFTSYHIHWV RQAPGQGLEWMGWIYPGNVNTEYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCAREEITYAMDY WGQGTTVTVSSGGGGSGGGGSGGGGSGGEIVLTQSPGTLSLSPGERATLSCRSSQSIVHSNGNTYLEWYQ QKPGQAPRLLIYKVSNRFSGIPDRFSGSGSGTPFTLTISRLEPEPFAVYYCFQGSHVPRTFGGGTKVEIK (SEQ ID NO: 36) C-002160QLVQSGAEVKKPGSSVKVSCKASGYTFTSYHIHWV RQAPGQGLEWMGWIYPGNVNTEYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCAREEITYAMDY WGQGTTVTVSSGGGGSGGGGSGGGGSGGDIVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYL QKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIK (SEQ ID NO: 37) C-002161QLVESGGGLVKPGGSLRLSCAASGYTFTSYHIHWV RQAPGKGLEWVGWIYPGNVNTEYNEKFKGRFTISRDDSKNTLYLQMNSLKTEDTAWYCAREEITYAMDYW GQGTTVTVSSGGGGSGGGGSGGGGSGGDIQMTQSPSSLSASVGDRVTITCRSSQSIVHSNGNTYLEWYQQ KPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSHVPRTFGGGTKVEIK (SEQ ID NO: 38) C-002162QLVQSGAEVKKPGSSVKVSCKASGYTFTSYHIHWV RQAPGQGLEWIGWIYPGNVNTEYNEKFKGKATITADESTNTAYMELSSLRSEDTAVYYCAREEITYAMDY WGQGTLVTVSSGGGGSGGGGSGGGGSGGDIQMTQSPSTLSASVGDRVTITCRSSQSIVIISNGNTYLEWY QQKPGKAPKLLIYKVSNRFSGVPARFSGSGSGTEFTLTISSLQPDDFATYYCFQGSHVPRTFGQGTKVEV K (SEQ ID NO: 39) C-002163QLVQSGAEVKKPGSSVKVSCKASGYTFTSYHMHWV RQAPGQGLEWIGYIYPGNVNTEYNEKFKGKATLTADKSTNTAYMELSSLRSEDTAVYFCAREEFITAMDY WGQGTLVTVSSGGGGSGGGGSGGGGSGGDVQMTQSPSTLSASVGPRVTITCSSSQSIVHSNGNTYMEWYQ QKPGKAPKLLIYKVSNRFSGVPDRFSGSGSGTEFTLTISSLQPDDFATYYCHQGSHVTRTFGQGTKVEVK (SEQ ID NO: 40) C-002164QVQLQQSGPELVKPGASVKMSCKASGYTFTSYHIQ WVKQRPGQGLEWIGWIYPGPGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLTSEDSAIYFCAREGTYYAM DYWGQGTSVTVSSGGGGSGGGGSGGGGSGGDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHSNGNTYLEW YLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLE TK(SEQ ID NO: 41) C-002165QLQLQESGPGLVKPSETLSLTCTVSGYTFTSYHIQ WIRQPPGKGLEWIGWIYPGDGSTQYNEKFKGRATISVDTSKNQFSLNLDSVSAADTAIYYCAREGTYYAM DYWGKGSTVTVSSGGGGSGGGGSGGGGSGGDIQMTQSPSSLSASVGDRVTITCRSSQSIVHSNGNTYLEW YQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQGSHVPRTFGPGTKVD DC(SEQ ID NO: 42) C-002166EVQLVQSGAELKKPGSSVKVSCKASGYTFTSYHIQ WVKQAPGQGLEWIGWIYPGPGSTQYNEKFKGKATLTVDKSTNTAYMELSSLRSEDTAVYYCAREGTYYAM DYWGQGTLVTVSSGGGGSGGGGSGGGGSGGDIQMTQSPSTLSASVGDRVTITCRSSQSIVHSNGNTYLEW YQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCFQGSHYPRTFGQGTKVE (SEQ ID NO: 43) WC-002167QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYHIQ WVRQAPGQGLEWMGWIYPGDGSTQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREGTYYAM DYWGQGTTVTVSSGGGGSGGGGSGGGGSGGEIVLTQSPGTLSLSPGERATLSCRSSQSIVHSNGNTYLEW YQQKPGQAPRLLIYKVSNRFSGIPDRFSGSGSGTDFTLTISRLEPFDFAVYYCFQGSHVPRTFGGGTKVE IK (SEQ ID NO: 44) C-002168QVTLKQSGAEVKKPGSSVKVSCTASGYTFTSYHVS WVRQAPGQGLEWLGRIYPGDGSTQYNEKFKGKVTITADKSMPTSFMELTSLTSEPTAVYYCAREGTYYAM DLWGQGTLVTVSSGGGGSGGGGSGGGGSGGEIVLTQSPGTLSLSPGERATLSCRSSQSIVHSNGNTYLAW YQQKPGQAPRLLISKVSNRFSGVPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGSHVPRTFGGGTKVE IK (SEQ ID NO: 45) C-002169QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHMH WVRQAPGQRLEWMGWIYPGDGSTQYNEKFKGKVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAM DYWGQGTLVTVSSGGGGSGGGGSGGGGSGGDIVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLDW YLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTPFTLKISRVEAEDVGVYYCMQGSHVPRTFGGGTKVE IK (SEQ ID NO: 46) CDR- CDR- CDR-CDR- CDR- CDR- L1 L2 L3 H1 H2 H3 RSSQ KVSN FQGS ASGY WIYP EEIT SIVH RFSGHVPR TFTS GNVN YAMD SNGN VPDR T YHIH TEYN Y TYLE (SEQ (SEQ (SEQ EKFK(SEQ (SEQ ID ID ID GK ID ID NO: NO: NO: (SEQ NO: NO: 23) 24) 25) ID 27)22) NO: 26) RSSQ KVSN MQGS SGYT WIYP EGTY SIVH RFSG HVPR FTSY GDGS YAMDSNGN VPDR T HMH TQYN Y TYLD (SEQ (SEQ (SEQ EKFK (SEQ (SEQ ID ID ID G IDID NO: NO: NO: (SEQ NO: NO: 29) 30) 31) ID 33) 28) NO: 32

In some embodiments, the scFv comprises the complementarity determinedregions (CDRs) of any one of SEQ ID NOS: 22-33. In some embodiments, thescFv comprises a sequence at least 95% identical to any one of SEQ IDNOS: 22-33. In some embodiments, the scFv comprises a sequence identicalto any one of SEQ ID NOS: 22-33. In some embodiments, the heavy chain ofthe antibody comprises the heavy chain CDRs of any one of SEQ ID NOS:25-27 or 31-33, and the light chain of the antibody comprises the lightchain CDRs of any one of SEQ ID NOS: 22-24 or 28-30. In someembodiments, the heavy chain of the antibody comprises a sequence atleast 95% identical to the heavy chain portion of any one of SEQ ID NOS:35-46 or 125, and wherein the light chain of the antibody comprises asequence at least 95% identical to the light chain portion of any one ofSEQ ID NOS: 35-46 or 125. In some embodiments, the heavy chain comprisesall of SEQ ID NOS: 25-27, and the light chain comprises all of SEQ IDNOS: 22-24. In some embodiments, the heavy chain comprises all of SEQ IDNOS: 31-33, and the light chain comprises all of SEQ ID NOS: 28-30.

In some embodiments, the heavy chain of the antibody comprises asequence identical to the heavy chain portion of any one of SEQ ID NOS:35-46 or 125, and wherein the light chain of the antibody comprises asequence identical to the light chain portion of any one of SEQ ID NOS:35-46 or 125.

In some embodiments, the ScFv comprises a sequence at least 95%identical, at least 96% identical, at least 97% identical, at least 98%identical, at least 99% identical or identical to any one of SEQ ID NOS:35-46 or 125.

B- and T-Lymphocyte Attenuator (BTLA) Domains

In some embodiments, the polypeptide comprises a B- and T-lymphocyteattenuator (BTLA) hinge domain, transmembrane domain, intracellulardomain or a functional variant, derivative or combination thereof.

In some embodiments, the polypeptide comprises a BTLA intracellulardomain. In some embodiments, the BTLA intracellular domain comprises asequence of RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS (SEQ ID NO: 87). Insome embodiments, the BTLA intracellular domain comprises SEQ ID NO: 87,or a sequence with at least 95% identity thereto. In some embodiments,the BTLA intracellular domain consists essentially SEQ ID NO: 87.

In some embodiments, the BTLA transmembrane domain and intracellulardomain comprises a sequence at least 95% identical to a sequence ofLLPLGGLPLLITTCFCLFCCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS (SEQ ID NO: 88). In some embodiments, the BTLAtransmembrane domain and intracellular domain comprises or consistsessentially of a sequence of SEQ ID NO: 88.

Signal Peptides

In some embodiments, the polypeptide comprises a signal peptide. Forexample, the polypeptide comprises a VK1 signal peptide. In someembodiments, the signal peptide is an N-terminal signal peptide. In someembodiments, the signal peptide comprises a sequence at least 95%identical to a sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 128). Insome embodiments, the signal peptide comprises a sequence ofMDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 128). In some embodiments, the signalpeptide is encoded by a sequence at least 95% identical to a sequence ofATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGCCAGATGT (SEQID NO: 129), or a sequence identical thereto.

Antigens

The skilled artisan will understand that any macromolecule, includingvirtually all proteins or peptides, can serve as an antigen for theLILRB1-based receptors described herein. Furthermore, antigens can bederived from recombinant or genomic DNA. A skilled artisan willunderstand that any DNA, which comprises a nucleotide sequences or apartial nucleotide sequence encoding a protein that elicits an immuneresponse therefore encodes an “antigen” as that term is used herein.Furthermore, one skilled in the art will understand that an antigen neednot be encoded solely by a full length nucleotide sequence of a gene.Moreover, a skilled artisan will understand that an antigen need not beencoded by a “gene” at all. It is readily apparent that an antigen canbe generated synthesized or can be derived from a biological sample, ormight be macromolecule besides a polypeptide. Such a biological samplecan include, but is not limited to a tissue sample, a tumor sample, acell or a fluid with other biological components.

In some embodiments, the antigen-binding domain specifically binds to atarget selected from etiolate receptor, αvββ integrin, TNF receptorsuperfamily member 17 (BCMA), CD276 molecule (B7-H3), natural killercell cytotoxicity receptor 3 ligand 1 (B7-H6), carbonic anhydrase 9(CAIX), CD19 molecule (CD19), membrane spanning 4-domains A1 (CD20),CD22 molecule (CD22), TNF receptor superfamily member 8 (CD30), CD33molecule (CD33), CD37 molecule (CD37), CD44 molecule (CD44), CD44v6,CD44v7/8, CD70 molecule (CD70), interleukin 3 receptor subunit alpha(CD123), syndecan 1 (CD138), L1 cell adhesion molecule (CD171), CEA celladhesion molecule (CEA), delta like canonical Notch ligand 4 (DLL4),epithelial cell adhesion molecule (EGP-2), epithelial cell adhesionmolecule (EGP-40), chondroitin sulfate proteoglycan 4 (CSPG4), epidermalgrowth factor receptor (EGFR), EGFR family including ErbB2 (HER2),EGFRvIII, epithelial cell adhesion molecule (EPCAM), EPH receptor A2(EphA2), EpCAM, fibroblast activation protein alpha (FAP), folatereceptor alpha (FBP), fetal acetylcholine receptor, frizzled classreceptor 7 (Fzd7), diganglioside GD2 (GD2), ganglioside GD3 (GD3),Glypican-3 (GPC3), trophoblast glycoprotein (h5T4), interleukin 11receptor subunit alpha (IL-1 IR), interleukin 13 receptor subunit alpha2 (IL13R-a2), kinase insert domain receptor (KDR), κ light chain, λlight chain, LeY, L1 cell adhesion molecule (L1 CAM), MAGE-A1,mesothelin, MHC presented peptides, mucin 1, cell surface associated(MUC1), mucin 16, cell surface associated (MUC16), neural cell adhesionmolecule 1 (NCAM), killer cell lectin like receptor K1 (NKG2D) ligands,Notch1, Notch2/3, NY-ESO-1, PRAME nuclear receptor transcriptionalregulator (PRAME), prostate stem cell antigen (PSCA), folate hydrolase 1(PSMA), Survivin, TAG-72, TEMs, telomerase reverse transcriptase (TERT),kinase insert domain receptor (VEGFR2), and receptor tyrosine kinaselike orphan receptor 1 (ROR1).

In some embodiments, the antigen-binding domain specifically binds to atarget selected from CD33, CD38, a human leukocyte antigen (HLA), anorgan specific antigen, a blood-brain barrier specific antigen, anEpithelial-mesenchymal transition (EMT) antigen, E-cadherin,cytokeratin, Opioid-binding protein/cell adhesion molecule (OPCML),HYLA2, Deleted in Colorectal Carcinoma (DCC), Scaffold/Matrix attachmentregion-binding protein 1 (SMAR1), cell surface carbohydrate and mucintype 0-glycan.

In some embodiments, the extracellular domain of the LILRB1-basedreceptors described herein comprises an antigen-binding domain specificto an antigen that is lost through loss of heterozygosity in cells of asubject.

As used herein, “loss of heterozygosity (LOH)” refers to a geneticchange that occurs at high frequency in cancers, whereby one of the twoalleles is deleted, leaving a single mono-allelic (hemizygous) locus.

In some embodiments, the LILRB1-based receptor comprises anantigen-binding domain specific to a minor histocompatibility antigen(MiHA). MiHAs are peptides derived from proteins that containnonsynonymous differences between alleles and are displayed by commonHLA alleles. The non-synonymous differences can arise from SNPs,deletions, frameshift mutations or insertions in the coding sequence ofthe gene encoding the MiHA. Exemplary MiHAs can be about 9-12 aminoacids in length and can bind to MHC class I and MHC class II proteins.Binding of the TCR to the MHC complex displaying the MiHA can activate Tcells. The genetic and immunological properties of MiHAs will be knownto the person of ordinary skill in the art, and specific MiHas describedin PCT/US2020/045228, the contents of which are incorporated byreference.

In some embodiments, the LILRB1-based receptor comprises anantigen-binding domain specific to an antigen that is lost in cancercells of a subject through loss of Y chromosome.

In some embodiments, the LILRB1-based receptor comprises anantigen-binding domain specific to an HLA class I allele. The majorhistocompatibility complex (MHC) class I is a protein complex thatdisplays antigens to cells of the immune system, triggering immuneresponse. The Human Leukocyte Antigens (HLAs) corresponding to MHC classI are HLA-A, HLA-B and HLA-C. HLA-E is known in the art as anon-classical MHC class I molecule. In some embodiments, the antigen forthe LILR1-based receptor comprises an HLA class I allele. In someembodiments, allele of HLA class I is lost in a target cell, such as acancer cell, through loss of heterozygosity (LOH).

HLA-A is a group of human leukocyte antigens (HLA) of the majorhistocompatibility complex (MHC) that are encoded by the HLA-A locus.HLA-A is one of three major types of human MHC class I cell surfacereceptors. The receptor is a heterodimer comprising a heavy α chain andsmaller β chain. The α chain is encoded by a variant of HLA-A, while theβ chain (β2-microglobulin) is invariant. There are several thousandHLA-A variants, all of which fall within the scope of the instantdisclosure.

In some embodiments, the LILRB1-based receptor comprises anantigen-binding domain specific to an HLA-B allele. The HLA-B gene hasmany possible variations (alleles). Hundreds of versions (alleles) ofthe HLA-B gene are known, each of which is given a particular number(such as HLA-B27).

In some embodiments, the LILRB1-based receptor comprises anantigen-binding domain specific to an HLA-C allele. HLA-C belongs to theHLA class I heavy chain paralogues. This class I molecule is aheterodimer consisting of a heavy chain and a light chain (beta-2microglobulin).

In some embodiments, the HLA class I allele has broad or ubiquitous RNAexpression.

In some embodiments, the HLA class I allele has a known, or generallyhigh minor allele frequency.

In some embodiments, the HLA class I allele does not require apeptide-MHC antigen, for example when the HLA class I allele isrecognized by a pan-HLA ligand binding domain.

In some embodiments, the LILRB1-based receptor comprises anantigen-binding domain specific to an HLA-A allele. In some embodimentsthe HLA-A allele comprises HLA-A*02. Various single variable domainsknown in the art or disclosed herein that bind to and recognize HLA-A*02are suitable for use in embodiments, and are described herein.

In some embodiments, the antigen-binding domain specifically binds to anHLA-A*02 antigen. In some embodiments, the antigen-binding domainspecifically binds to an HLA-A*02 antigen in a peptide-independentmanner.

Polynucleotides and Vectors

In other aspects, the disclosure provides polynucleotides comprising anucleic acid sequence encoding receptors of the disclosure. In someembodiments, the polynucleotides encode one or more of an LILRB1 hingedomain, an LILRB1 transmembrane domain and an LILRB1 intracellulardomain or a functional derivative or fragment thereof.

In some embodiments, the polynucleotide comprises a nucleic acidsequence that encodes a polypeptide that is at least 95% identical toany one of SEQ ID NOS: 1-7 or 12-21. In some embodiments, thepolynucleotide comprises a nucleic acid sequence that encodes apolypeptide that is at least 95% identical to any one of SEQ ID NOS:47-71, 77-79, 89-92, 120 or 122. In some embodiments, the polynucleotidecomprises a nucleic acid sequence that encodes a polypeptide that is atleast 95% identical to the heavy chain portion or the light chainportion of any one of SEQ ID NOS: 35-46 or 125. In some embodiments, thepolynucleotide comprises a nucleic acid sequence that encodes apolypeptide that is at least 95% identical to the heavy chain portion orthe light chain portion of any one of SEQ ID NOS. 35, 39, 46 or 125. Insome embodiments, the polynucleotide comprises a nucleic acid sequencethat encodes a polypeptide that is identical to the heavy chain portionor the light chain portion of any one of SEQ ID NOS: 35, 39, 46 or 125.In another aspect, the disclosure provides vectors comprising thepolynucleotides encoding receptors of the disclosure.

In some embodiments, the polynucleotide comprises a sequence at least95% identical to SEQ ID NO: 121 or 123. In some embodiments, thepolynucleotide comprises SEQ ID NO: 121 or 123.

In some embodiments, the polynucleotide comprises a sequence of a LILRB1hinge, transmembrane and intracellular domain. In some embodiments, thepolynucleotide comprises a sequence at least 95% identical to SEQ ID NO:126. In some embodiments, the polynucleotide comprises a sequence of SEQID NO: 126.

Vectors derived from retroviruses such as the lentivirus are suitabletools to achieve long-term gene transfer since they allow long-term,stable integration of a transgene and its propagation in daughter cells.Lentiviral vectors have the added advantage over vectors derived fromonco-retroviruses such as murine leukemia viruses in that they cantransduce non-proliferating cells, such as hepatocytes. They also havethe added advantage of low immunogenicity.

The expression of natural or synthetic nucleic acids encoding receptorsis typically achieved by operably linking a nucleic acid encodingreceptor or portions thereof to a promoter, and incorporating theconstruct into an expression vector. The vectors can be suitable forreplication and integration eukaryotes. Typical cloning vectors containtranscription and translation terminators, initiation sequences, andpromoters useful for regulation of the expression of the desired nucleicacid sequence.

The polynucleotides encoding the receptors can be cloned into a numberof types of vectors. For example, the polynucleotides can be cloned intoa vector including, but not limited to a plasmid, a phagemid, a phagederivative, an animal virus, and a cosmid. Vectors of particularinterest include expression vectors, replication vectors, probegeneration vectors, and sequencing vectors.

Further, the expression vector may be provided to cells, such as immunecells, in the form of a viral vector. Viral vector technology is wellknown in the art and is described, for example, in Sambrook et al.(2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York), and in other virology and molecular biologymanuals. Viruses, which are useful as vectors include, but are notlimited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, a suitable vector contains anorigin of replication functional in at least one organism, a promotersequence, convenient restriction endonuclease sites, and one or moreselectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

In some embodiments, the vector comprises a promoter. Vectors can alsoinclude additional regulatory elements. Additional regulatory elements,e.g., enhancers, regulate the frequency of transcriptional initiation.Typically, these are located in the region 30-110 basepairs (bp)upstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the thymidine kinase (tk) promoter,the spacing between promoter elements can be increased to 50 bp apartbefore activity begins to decline. Depending on the promoter, it appearsthat individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess the expression of receptor the expression vector tobe introduced into a cell can also contain either a selectable markergene or a reporter gene or both to facilitate identification andselection of expressing cells from the population of cells sought to betransfected or infected through viral vectors. In other aspects, theselectable marker may be carried on a separate piece of DNA and used ina co-transfection procedure. Both selectable markers and reporter genesmay be flanked with appropriate regulatory sequences to enableexpression in the host cells. Useful selectable markers include, forexample, antibiotic-resistance genes, such as neo and the like.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). One method for the introduction of a polynucleotide into ahost cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virus1, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

Engineered Cells

In another aspect, the disclosure provides immune cells comprising anucleic acid sequence or vector encoding receptors of the disclosureand/or expressing receptors of the disclosure.

In embodiments, immune cell activation is reduced when the cell iscontacted with the antigen of the LILRB1 based receptors of thedisclosure, or a cell expressing the antigen on its surface. Inembodiments, immune cell activation comprises expression of a geneoperatively linked to an NFAT promoter. Immune cell activation and/orinhibition of activation can be measured by various other methods knownin the art. In some embodiments, the immune cell comprises an additionalexogenous receptor, for example an activator receptor such as a chimericantigen receptor (CAR) or TCR.

In embodiments, the immune cell is a T cell.

As used herein, the term “immune cell” refers to a cell involved in theinnate or adaptive (acquired) immune systems. Exemplary innate immunecells include phagocytic cells such as neutrophils, monocytes andmacrophages, Natural Killer (NK) cells, polymophonuclear leukocytes suchas neutrophils eosinophils and basophils and mononuclear cells such asmonocytes, macrophages and mast cells. Immune cells with roles inacquired immunity include lymphocytes such as T-cells and B-cells.

As used herein, a “T-cell” refers to a type of lymphocyte thatoriginates from a bone marrow precursor that develops in the thymusgland. There are several distinct types of T-cells which develop uponmigration to the thymus, which include, helper CD4+ T-cells, cytotoxicCD8+ T cells, memory T cells, regulatory CD4+ T-cells and stem memoryT-cells. Different types of T-cells can be distinguished by theordinarily skilled artisan based on their expression of markers. Methodsof distinguishing between T-cell types will be readily apparent to theordinarily skilled artisan.

Method of Making Engineered Cells

In another aspect, the disclosure provides methods comprisingintroducing a polynucleotide of the disclosure into cells, optionallyusing vectors of the disclosure. In embodiments, the resulting cellexpresses LILRB1 based receptor encoded by the polynucleotide. Inembodiments, the cell is an immune cell. In embodiments, the immune cellis a T cell.

Methods of transforming populations of immune cells, such as T cells,with the vectors of the instant disclosure will be readily apparent tothe person of ordinary skill in the art. For example, CD3+ T cells canbe isolated from PBMCs using a CD3+ T cell negative isolation kit(Miltenyi), according to manufacturer's instructions. T cells can becultured at a density of 1×10{circumflex over ( )}6 cells/mL in X-Vivo15 media supplemented with 5% human A/B serum and 1% Pen/strep in thepresence of CD3/28 Dynabeads (1:1 cell to bead ratio) and 300 Units/mLof IL-2 (Miltenyi). After 2 days, T cells can be transduced with viralvectors, such as lentiviral vectors using methods known in the art. Insome embodiments, the viral vector is transduced at a multiplicity ofinfection (MOI) of 5. Cells can then be cultured in IL-2 or othercytokines such as combinations of IL-7/15/21 for an additional 5 daysprior to enrichment. Methods of isolating and culturing otherpopulations of immune cells, such as B cells, or other populations of Tcells, will be readily apparent to the person of ordinary skill in theart. Although this method outlines a potential approach it should benoted that these methodologies are rapidly evolving. For exampleexcellent viral transduction of peripheral blood mononuclear cells canbe achieved after 5 days of growth to generate a >99% CD3+ highlytransduced cell population.

Methods of activating and culturing populations of T cells comprisingthe receptors, polynucleotides or vectors of the disclosure will bereadily apparent to the person of ordinary skill in the art.

Whether prior to or after genetic modification, T cells can be activatedand expanded generally using methods as described, for example, in U.S.Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358;6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566;7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041, 10,040,846; andU.S. Pat. Appl. Pub. No. 2006/0121005.

In some embodiments, T cells of the instant disclosure are expanded andactivated in vitro. Generally, the T cells of the instant disclosure areexpanded in vitro by contact with a surface having attached thereto anagent that stimulates a CD3/TCR complex associated signal and a ligandthat stimulates a co-stimulatory molecule on the surface of the T cells.In particular, T cell populations may be stimulated as described herein,such as by contact with an anti-CD3 antibody. For co-stimulation of anaccessory molecule on the surface of the T cells, a ligand that bindsthe accessory molecule is used. For example, a population of T cells canbe contacted with an anti-CD3 antibody and an anti-CD28 antibody, underconditions appropriate for stimulating proliferation of the T cells. Tostimulate proliferation of either CD4+ T cells or CD8+ T cells, ananti-CD3 antibody and an anti-CD28 antibody can be used. Examples of ananti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besançon,France) can be used as can other methods commonly known in the art (Berget al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp.Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth.227(1-2):53-63, 1999).

In some embodiments, the primary stimulatory signal and theco-stimulatory signal for the T cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In someembodiments, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells in the present invention.

In some embodiments, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one embodiment, a 1:1ratio of each antibody bound to the beads for CD4+ T cell expansion andT cell growth is used. In some embodiments, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain embodiments ofthe invention, the ratio of anti CD28 antibody to anti CD3 antibodybound to the beads is greater than 2:1.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:9 to 9:1 andany integer values in between, can also be used to stimulate T cells. Insome embodiments, a ratio of 1:1 cells to beads is used. One of skill inthe art will appreciate that a variety of other ratios may be suitablefor use in the present invention. In particular, ratios will varydepending on particle size and on cell size and type.

In further embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached tocontact the T cells. In one embodiment the cells (for example, CD4+ Tcells) and beads (for example, DYNABEADS CD3/CD28 T paramagnetic beadsat a ratio of 1:1) are combined in a buffer. Again, those of ordinaryskill in the art can readily appreciate any cell concentration may beused. In certain embodiments, it may be desirable to significantlydecrease the volume in which particles and cells are mixed together(i.e., increase the concentration of cells), to ensure maximum contactof cells and particles. For example, in one embodiment, a concentrationof about 2 billion cells/ml is used. In another embodiment, greater than100 million cells/ml is used. In a further embodiment, a concentrationof cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml isused. In yet another embodiment, a concentration of cells from 75, 80,85, 90, 95, or 100 million cells/ml is used. In further embodiments,concentrations of 125 or 150 million cells/ml can be used. In someembodiments, cells that are cultured at a density of 1×10⁶ cells/mL areused.

In some embodiments, the mixture may be cultured for several hours(about 3 hours) to about 14 days or any hourly integer value in between.In another embodiment, the beads and T cells are cultured together for2-3 days. Conditions appropriate for T cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-vivo 15, (Lonza)) that may contain factors necessary for proliferationand viability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells. In someembodiments, the media comprises X-VIVO-15 media supplemented with 5%human A/B serum, 1% penicillin/streptomycin (pen/strep) and 300 Units/mlof IL-2 (Miltenyi).

The T cells are maintained under conditions necessary to support growth,for example, an appropriate temperature (e.g., 37° C.) and atmosphere(e.g., air plus 5% CO2).

In some embodiments, the T cells comprising receptors of the disclosureare autologous. Prior to expansion and genetic modification, a source ofT cells is obtained from a subject. Immune cells such as T cells can beobtained from a number of sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, and tumors. In certain embodiments of the presentinvention, any number of T cell lines available in the art, may be used.In certain embodiments of the present invention, T cells can be obtainedfrom a unit of blood collected from a subject using any number oftechniques known to the skilled artisan, such as Ficoll™ separation.

In some embodiments, cells from the circulating blood of an individualare obtained by apheresis. The apheresis product typically containslymphocytes, including T cells, monocytes, granulocytes, B cells, othernucleated white blood cells, red blood cells, and platelets. In someembodiments, the cells collected by apheresis may be washed to removethe plasma fraction and to place the cells in an appropriate buffer ormedia for subsequent processing steps. In some embodiments, the cellsare washed with phosphate buffered saline (PBS). In alternativeembodiments, the wash solution lacks calcium and may lack magnesium ormay lack many if not all divalent cations. As those of ordinary skill inthe art would readily appreciate a washing step may be accomplished bymethods known to those in the art, such as by using a semi-automated“flow-through” centrifuge (for example, the Cobe 2991 cell processor,the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to themanufacturer's instructions. After washing, the cells may be resuspendedin a variety of biocompatible buffers, such as, for example, Ca2+-free,Mg2+-free PBS, PlasmaLyte A, or other saline solution with or withoutbuffer. Alternatively, the undesirable components of the apheresissample may be removed and the cells directly resuspended in culturemedia.

In some embodiments, immune cells such as T cells are isolated fromperipheral blood lymphocytes by lysing the red blood cells and depletingthe monocytes, for example, by centrifugation through a PERCOLL™gradient or by counterflow centrifugal elutriation. Specificsubpopulations of immune cells, such as T cells, B cells, or CD4+ Tcells can be further isolated by positive or negative selectiontechniques. For example, in one embodiment, T cells are isolated byincubation with anti-CD4-conjugated beads, for a time period sufficientfor positive selection of the desired T cells.

Enrichment of an immune cell population, such as a T cell population, bynegative selection can be accomplished with a combination of antibodiesdirected to surface markers unique to the negatively selected cells. Onemethod is cell sorting and/or selection via negative magneticimmune-adherence or flow cytometry that uses a cocktail of monoclonalantibodies directed to cell surface markers present on the cellsnegatively selected. For example, to enrich for CD4+ cells by negativeselection, a monoclonal antibody cocktail typically includes antibodiesto CD 14, CD20, CD 11b, CD 16, HLA-DR, and CD8.

For isolation of a desired population of immune cells by positive ornegative selection, the concentration of cells and surface (e.g.,particles such as beads) can be varied. In certain embodiments, it maybe desirable to significantly decrease the volume in which beads andcells are mixed together (i.e., increase the concentration of cells), toensure maximum contact of cells and beads.

In some embodiments, the cells may be incubated on a rotator for varyinglengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation, or PBMCs from which immune cells such as Tcells are isolated, can also be frozen after a washing step. Wishing notto be bound by theory, the freeze and subsequent thaw step provides amore uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

Assaying Signaling

In some embodiments, immune cell activation is reduced when the cell iscontacted with the antigen corresponding to the LILRB1 based receptor ofthe disclosure, or a cell expressing the antigen on its surface. In someembodiments, immune cell activation comprises expression of a geneoperatively linked to an NFAT promoter. Nuclear factor of activatedT-cells (NFAT) is a family of transcription factors shown to beimportant in immune response. The NFAT transcription factor familyconsists of five members NFATc1, NFATc2, NFATc3, NFATc4, and NFAT5. NFATplays a role in regulating inflammation.

As used herein, an NFAT promoter is a promoter that is regulated (i.e.,activated or repressed) when NFAT is expressed in a cell. NFAT targetpromoters are described in Badran, B. M. et al. (2002) J. BiologicalChemistry Vol. 277: 47136-47148, and contain NFAT consensus sequencessuch as GGAAA.

Methods of assessing the effects of receptor activation on geneexpression are known in the art, and include the use of reporter genes,whose expression can be quantified. Reporter genes are used foridentifying potentially transfected or transduced cells and forevaluating the functionality of regulatory sequences. In general, areporter gene is a gene that is not present in or expressed by therecipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription. In exemplary embodiments, an NFATpromoter operably linked to a reporter gene is used to evaluate theexpression of the receptors of the disclosure on NFAT signaling.

Pharmaceutical Compositions

The disclosure provides pharmaceutical compositions comprising immunecells comprising the LILRB1-based receptors of the disclosure apharmaceutically acceptable diluent, carrier or excipient.

Such compositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; and preservatives.

Methods of Treating Disease

Provided herein are methods of treating a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of a composition comprising a plurality of immune cellscomprising the LILRB1-based receptors described herein. In someembodiments, the immune cells further comprise an activator receptor,such as an activator CAR or TCR.

Additional methods of treating subjects, and activator receptorscombination combined with inhibitory receptors, are described inPCT/US2020/045228, the contents of which are incorporated by referenceherein in their entirety.

In some embodiments, the subject in need thereof has cancer. In someembodiments, the methods of treating the subject comprise administeringto the subject a plurality of immune cells comprising theLILRB1-receptors of the disclosure. In some embodiments, the pluralityof immune cells further comprises an activator receptor, such as a CARor a TCR. In some embodiments, the CAR or TCR comprises anantigen-binding domain specific to a cancer antigen. Activator receptorsspecific for cancer antigens can comprise antigen-binding domainsisolated or derived from any antibody or antigen-binding domain known inthe art, including, but not limited to, urelumab, utomilumab, oleclumab,naptumomab, ascrinvacumab, tacatuzumab, nesvacumab, vanucizumab,belimumab, tabalumab, tibulizumab, belantamab, igovomab, oregovomab,sofituzumab, mogamulizumab, talacotuzumab, tavolimab, vonlerolizumab,ipilimumab, duvortuxizumab, blinatumomab, coltuximab, denintuzumab,inebilizumab, loncastuximab, taplitumomab, ibritumomab, obinutuzumab,ocaratuzumab, ocrelizumab, ofatumumab, rituximab, tositumomab,veltuzumab, samalizumab, bectumomab, epratuzumab, inotuzumab,moxetumomab, pinatuzumab, gomiliximab, lumiliximab, camidanlumab,basiliximab, inolimomab, daclizumab, varlilumab, enoblituzumab,omburtamab, brentuximab, iratumumab, gemtuzumab, lintuzumab,vadastuximab, lilotomab, otlertuzumab, tetulomab, daratumumab,isatuximab, bivatuzumab, abituzumab, intetumumab, lorvotuzumab,itolizumab, cusatuzumab, vorsetuzumab, milatuzumab, polatuzumab,iladatuzumab, galixima, altumomab, arcitumomab, labetuzumab,cibisatamab, zolbetuximab, lacnotuzumab, cabiralizumab, emactuzumab,gimsilumab, lenzilumab, otilimab, mavrilimumab, tremelimumab,ulocuplumab, tepoditamab, rovalpituzumab, demcizumab, drozitumab,parsatuzumab, cetuximab, depatuxizumab, futuximab, imgatuzumab,laprituximab, matuzumab, necitumumab, nimotuzumab, panitumumab,zalutumumab, modotuximab, amivantamab, tomuzotuximab, losatuxizumab,adecatumumab, citatuzumab, edrecolomab, oportuzumab, solitomab,tucotuzumab, catumaxomab, ifabotuzumab, duligotuzumab, elgemtumab,lumretuzumab, patritumab, seribantumab, zenocutuzumab, aprutumab,bemarituzumab, vantictumab, dinutuximab, ecromeximab, mitumomab,codrituzumab, glembatumumab, zatuximab, ertumaxomab, margetuximab,timigutuzumab, gancotamab, pertuzumab, trastuzumab, ficlatuzumab,rilotumumab, telisotuzumab, emibetuzumab, cixutumumab, dalotuzumab,figitumumab, ganitumab, robatumumab, teprotumumab, flotetuzumab,bermekimab, cergutuzumab, volociximab, etaracizumab, relatlimab,carlumab, amatuximab, clivatuzumab, gatipotuzumab, pemtumomab,cantuzumab, pankomab, racotumomab, brontictuzumab, tarextumabmvesencumab, camrelizumab, cetrelimab, nivolumab, pembrolizumab,pidilizumab, cemiplimab, spartalizumab, atezolizumab, avelumab,durvalumab, cirmtuzumab, tenatumomab, fresolimumab, brolucizumab,bevacizumab, ranibizumab, varisacumab, faricimab, icrucumab, alacizumab,and ramucirumab.

In some embodiments, the LILRB1-based receptor of the disclosurecomprises an antigen-binding domain specific to an antigen that is lostin the cancer cells through loss of heterozygosity. In some embodiments,the antigen is a minor histocompatibility antigen (MiHA). In someembodiments, the antigen is an HLA class I allele. In some embodiments,the HLA class I allele comprises HLA-A, HLA-B or HLA-C. In someembodiments, the HLA class I allele comprises HLA-E. In someembodiments, the HLA class I allele is an HLA-A*02 allele. In someembodiments, the antigen is not expressed in the target cell due to lossof Y chromosome. In some embodiments, the antigen specific to theLILRB1-based receptor is an HLA-A*02 antigen.

In some embodiments, the subject in need thereof has cancer. Cancer is adisease in which abnormal cells divide without control and spread tonearby tissue. In some embodiments, the cancer comprises a liquid tumoror a solid tumor. Exemplary liquid tumors include leukemias andlymphomas. Further cancers that are liquid tumors can be those thatoccur, for example, in blood, bone marrow, and lymph nodes, and caninclude, for example, leukemia, myeloid leukemia, lymphocytic leukemia,lymphoma, Hodgkin's lymphoma, melanoma, and multiple myeloma. Leukemiasinclude, for example, acute lymphoblastic leukemia (ALL), acute myeloidleukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenousleukemia (CML), and hairy cell leukemia. Exemplary solid tumors includesarcomas and carcinomas. Cancers can arise in virtually an organ in thebody, including blood, bone marrow, lung, breast, colon, bone, centralnervous system, pancreas, prostate and ovary. Further cancers that aresolid tumors include, for example, prostate cancer, testicular cancer,breast cancer, brain cancer, pancreatic cancer, colon cancer, thyroidcancer, stomach cancer, lung cancer, ovarian cancer, Kaposi's sarcoma,skin cancer, squamous cell skin cancer, renal cancer, head and neckcancers, throat cancer, squamous carcinomas that form on the moistmucosal linings of the nose, mouth, throat, bladder cancer,osteosarcoma, cervical cancer, endometrial cancer, esophageal cancer,liver cancer, and kidney cancer. In some embodiments, the conditiontreated by the methods described herein is metastasis of melanoma cells,prostate cancer cells, testicular cancer cells, breast cancer cells,brain cancer cells, pancreatic cancer cells, colon cancer cells, thyroidcancer cells, stomach cancer cells, lung cancer cells, ovarian cancercells, Kaposi's sarcoma cells, skin cancer cells, renal cancer cells,head or neck cancer cells, throat cancer cells, squamous carcinomacells, bladder cancer cells, osteosarcoma cells, cervical cancer cells,endometrial cancer cells, esophageal cancer cells, liver cancer cells,or kidney cancer cells.

Any cancer wherein a plurality of the cancer cells express the first,activator ligand and do not express the second, inhibitor ligand isenvisaged as within the scope of the instant disclosure. For example,CEA positive cancers that can be treated using the methods describedherein include colorectal cancer, pancreatic cancer, esophageal cancer,gastric cancer, lung adenocarcinoma, head and neck cancer, diffuse largeB cell cancer or acute myeloid leukemia cancer.

Treating cancer can result in a reduction in size of a tumor. Areduction in size of a tumor may also be referred to as “tumorregression”. Preferably, after treatment, tumor size is reduced by 5% orgreater relative to its size prior to treatment; more preferably, tumorsize is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75% or greater.Size of a tumor may be measured by any reproducible means ofmeasurement. The size of a tumor may be measured as a diameter of thetumor.

Treating cancer can result in a reduction in tumor volume. Preferably,after treatment, tumor volume is reduced by 5% or greater relative toits size prior to treatment; more preferably, tumor volume is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75% or greater. Tumor volume may bemeasured by any reproducible means of measurement.

Treating cancer results in a decrease in number of tumors. Preferably,after treatment, tumor number is reduced by 5% or greater relative tonumber prior to treatment; more preferably, tumor number is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75%. Number of tumors may bemeasured by any reproducible means of measurement. The number of tumorsmay be measured by counting tumors visible to the naked eye or at aspecified magnification. Preferably, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in a decrease in number of metastatic lesionsin other tissues or organs distant from the primary tumor site.Preferably, after treatment, the number of metastatic lesions is reducedby 5% or greater relative to number prior to treatment; more preferably,the number of metastatic lesions is reduced by 10% or greater; morepreferably, reduced by 20% or greater; more preferably, reduced by 30%or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75%. The number of metastatic lesions may be measured byany reproducible means of measurement. The number of metastatic lesionsmay be measured by counting metastatic lesions visible to the naked eyeor at a specified magnification. Preferably, the specified magnificationis 2×, 3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population receivingcarrier alone. Preferably, the average survival time is increased bymore than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population ofuntreated subjects. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in increase in average survival time of apopulation of treated subjects in comparison to a population receivingmonotherapy with a drug that is not a compound of the present invention,or a pharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in a decrease in the mortality rate of apopulation of treated subjects in comparison to a population receivingcarrier alone. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to an untreatedpopulation. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to a populationreceiving monotherapy with a drug that is not a compound of the presentinvention, or 1a pharmaceutically acceptable salt, prodrug, metabolite,analog or derivative thereof. Preferably, the mortality rate isdecreased by more than 2%; more preferably, by more than 5%; morepreferably, by more than 10%; and most preferably, by more than 25%. Adecrease in the mortality rate of a population of treated subjects maybe measured by any reproducible means. A decrease in the mortality rateof a population may be measured, for example, by calculating for apopulation the average number of disease-related deaths per unit timefollowing initiation of treatment with an active compound. A decrease inthe mortality rate of a population may also be measured, for example, bycalculating for a population the average number of disease-relateddeaths per unit time following completion of a first round of treatmentwith an active compound.

Treating cancer can result in a decrease in tumor growth rate.Preferably, after treatment, tumor growth rate is reduced by at least 5%relative to number prior to treatment; more preferably, tumor growthrate is reduced by at least 10%; more preferably, reduced by at least20%; more preferably, reduced by at least 30%; more preferably, reducedby at least 40%; more preferably, reduced by at least 50%; even morepreferably, reduced by at least 50%; and most preferably, reduced by atleast 75%. Tumor growth rate may be measured by any reproducible meansof measurement. Tumor growth rate can be measured according to a changein tumor diameter per unit time.

Treating cancer can result in a decrease in tumor regrowth. Preferably,after treatment, tumor regrowth is less than 5%; more preferably, tumorregrowth is less than 10%; more preferably, less than 20%; morepreferably, less than 30%; more preferably, less than 40%; morepreferably, less than 50%; even more preferably, less than 50%; and mostpreferably, less than 75%. Tumor regrowth may be measured by anyreproducible means of measurement. Tumor regrowth is measured, forexample, by measuring an increase in the diameter of a tumor after aprior tumor shrinkage that followed treatment. A decrease in tumorregrowth is indicated by failure of tumors to reoccur after treatmenthas stopped.

Treating or preventing a cell proliferative disorder can result in areduction in the rate of cellular proliferation. Preferably, aftertreatment, the rate of cellular proliferation is reduced by at least 5%;more preferably, by at least 10%; more preferably, by at least 20%; morepreferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The rate of cellular proliferation maybe measured by any reproducible means of measurement. The rate ofcellular proliferation is measured, for example, by measuring the numberof dividing cells in a tissue sample per unit time.

Treating or preventing a cell proliferative disorder can result in areduction in the proportion of proliferating cells. Preferably, aftertreatment, the proportion of proliferating cells is reduced by at least5%; more preferably, by at least 10%; more preferably, by at least 20%;more preferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The proportion of proliferating cellsmay be measured by any reproducible means of measurement. Preferably,the proportion of proliferating cells is measured, for example, byquantifying the number of dividing cells relative to the number ofnondividing cells in a tissue sample. The proportion of proliferatingcells can be equivalent to the mitotic index.

Treating or preventing a cell proliferative disorder can result in adecrease in size of an area or zone of cellular proliferation.Preferably, after treatment, size of an area or zone of cellularproliferation is reduced by at least 5% relative to its size prior totreatment; more preferably, reduced by at least 10%; more preferably,reduced by at least 20%; more preferably, reduced by at least 30%; morepreferably, reduced by at least 40%; more preferably, reduced by atleast 50%; even more preferably, reduced by at least 50%; and mostpreferably, reduced by at least 75%. Size of an area or zone of cellularproliferation may be measured by any reproducible means of measurement.The size of an area or zone of cellular proliferation may be measured asa diameter or width of an area or zone of cellular proliferation.

Treating or preventing a cell proliferative disorder can result in adecrease in the number or proportion of cells having an abnormalappearance or morphology. Preferably, after treatment, the number ofcells having an abnormal morphology is reduced by at least 5% relativeto its size prior to treatment; more preferably, reduced by at least10%; more preferably, reduced by at least 20%, more preferably, reducedby at least 30%; more preferably, reduced by at least 40%; morepreferably, reduced by at least 50%; even more preferably, reduced by atleast 50%; and most preferably, reduced by at least 75%. An abnormalcellular appearance or morphology may be measured by any reproduciblemeans of measurement. An abnormal cellular morphology can be measured bymicroscopy, e.g., using an inverted tissue culture microscope. Anabnormal cellular morphology can take the form of nuclear pleiomorphism.

Kits and Articles of Manufacture

The disclosure provides kits and articles of manufacture comprising thepolynucleotides and vectors encoding the receptors described herein. Insome embodiments, the kit comprises articles such as vials, syringes andinstructions for use.

In some embodiments, the kit comprises a polynucleotide or vectorcomprising a sequence encoding one or more chimeric antigen receptors ofthe disclosure. For example, the polynucleotide or vector comprises asequence one or more LILRB1 domains as described herein.

In some embodiments, the kit comprises a plurality of immune cellscomprising a chimeric antigen receptor as described herein. In someembodiments, the plurality of immune cells comprises a plurality of Tcells.

Polypeptide Sequences for Elements ofIllustrative Chimeric Antigen Receptors Name Sequence LILRB1MTPILTVLICLGLSL GPRTHVQAGHLPKPT LWAEPGSVITQGSPV TLRCQGGQETQEYRLYREKKTALWITRIPQ ELVKKGQFPIPSITW EHAGRYRCYYGSDTA GRSESSDPLELVVTGAYIKPTLSAQPSPVV NSGGNVILQCDSQVA FDGFSLCKEGEDEHP QCLNSQPHARGSSRAIFSVGPVSPSRRWWY RCYAYDSNSPYEWSL PSDLLELLVLGVSKK PSLSVQPGPIVAPEE TLTLQCGSDAGYNRFVLY KDGERDFLQLAGAQP QAGLSQANFTLGPVS RSYGGQYRCYGAHNLSSEWSAPSDPLDILI AGQFYDRVSLSVQPG PTVASGENVTLLCQS QGWMQTFLLTKEGAADDPWRLRSTYQSQKY QAEFPMGPVTSAHAG TYRC YGSQSSKPYLL THPSDPLEL VVSGPSGGPSSPTTGPTSTSG PEDQPLTPTGSDPQS GLGRHLGVVIGILVA VILLLLLLLLLFLILRHRRQGKHWTSTQRK ADFQHPAGAVGPEPT DRGLQWRSSPAADAQ EEN LYAAV KHTQPEDGVEMDTRSPHDEDPQ A VTYAEV KHSRPRRE MASPPSPLSGEFLDT KDRQAEEDRQMDTEAAASEAPQD VTYAQ LH SLTLRREATEPPPSQ EGPSPAVP SIYATL A IHPSQEGPSPAVPSIYATLAIH SEQ ID NO: 1 LILRB1 hinge- YGSQSSKPYLLTHPS transmembrane- DPLELVVSGPSGGPS intracelluiar domain SPITGPTSTSGPEDQ (SEQ ID NO: 126 is thePLTPTGSDPQSGLGR polynucleotide HLGVVIGILVAVILL sequence) LLLLLLLFLILRHRRQGKHWTSTQRKADFQ HPAGAVGPEPTDRGL QWRSSPAADAQEENL YAAVKHTQPEDGVEMDTRSPHDEDPQAVTY AEVKHSRPRREMASP PSPLSGEFLDTKDRQ AEEDRQMDTEAAASEAPQDVTYAQLHSLTL RREATEPPPSQEGPS PAVPSIYATLAIH SEQ ID NO: 2 LILRB1 hinge-VVSGPSGGPSSPTTG transmembrane- PTSTSGPEDQPLTPT intraceilularGSDPQSGLGRHLGVV domain (w/o IGILVAVILLLLLLL YGSQSSKPYLLTHPSDLLFLILRHRRQGKHW PLEL, TSTQRKADFQHPAGA SEQ ID NO: 18) VGPEPTDRGLQWRSSPAADAQEENLYAAVK HTQPEDGVEMDTRSP HDEDPQAVTYAEVKH SRPRREMASPPSPLSGEFLDTKDRQAEEDR QMDTEAAASEAPQDV TYAQLHSLTLRREAT EPPPSQEGPSPAVPS IYATLAIHSEQ ID NO: 3 LILRB1 hinge YGSQSSKPYLLTHPS domain DPLEL VVSGPSGGPSSPTTGPTSTSGPEDQP LTPTGSDPQSQLGRHL G SEQ ID NO: 4 LILRB1 VVIGILVAVILLLLLtransmembrane LLLLFLIL domain SEQ ID NO: 5 LILRB1 RHRRQGKHWTSTQRKintracellular ADFQHPAGAVGPEPT domain DRGLQWRSSPAADAQ EENLYAAVKHTQPEDGVEMDTRS PHDEDPQAVTYAEVK HSRPRREMASPPSPL SGEFLDTKDRQAEEDRQMDTEAAASEAPQD VTYAQLHSLTLRREA TEPPPSQEGPSPAVP SIYATLAIH SEQ ID NO: 7ITIM1 NLYAAV SEQ ID NO. 8 ITIM2 VTYAEV SEQ ID NO: 9 ITIM3 VTYAQLSEQ ID NO: 10 ITLM4 SIYATL SEQ ID NO: 11 ITIM 1-2 NLYAAV KHTQPEDGVEMDTRSPHDEDPQA V TYAEV SEQ ID NO: 12 ITIM2-3 VTYAEV KHSRPRREMASPPSPLSGEFLDTK DRQAEEDRQMDTEAA ASEAPQD VTYAQL SEQ ID NO: 13 ITIM3-4VTYAQL HSLTLRREA TEPPPSQEGPSPAVP SIYATL SEQ ID NO: 14 ITIM1-3 NLYAAVKHTQPEDGV EMDTRSPHDEDPQA V TYAEV KHSRPRREMA SPPSPLSGEFLDTKDRQAEEDRQMDTEAAA SEAPQDV TYAQL SEQ ID NO: 15 ITIM2-4 VTYAEV KHSRPRREMASPPSPLSGEFLDTK DRQAEEDRQMDTEAA ASEAPQDVTYAQLHS LTLRREATEPPPSQE GPSPAVPSIYATL SEQ ID NO: 16 ITIM1-4 NLYAAV KHTQPEDGV EMDTRSPHDEDPQA V TYAEVKHSRPRREMA SPPSPLSGEFLDTKD RQAEEDRQMDTEAAA SEA PQD VTYAQL HSLTLRREATEPPPSQEGPSP AVP SIYATL SEQ ID NO: 17 D3D4 domain YGSQSSKPYLLTHPSDPLEL SEQ ID NO: 18 Short hinge VVSGPSGGPSSPTTG PTSTSGPEDQPLTPTGSDPQSGLGRH LG SEQ ID NO: 19 Hinge (iTIM hinge) YGSQSSKPYLLTHPS DPLELVVSGPSGGPS SPTTGPTSTSGP EDQPLTPTGSDPQSG LGRHLGV (SEQ ID NO: 80)Short hinge 2 VVSGPSGGPSSPTTG PTSTSGPBDQPLTPT GSDPQSGLGRH LGV(SEQ ID NO: 81) Long hinge 1 AGSGGSGGSGGSPVP STPPTPSPSTPPTPSPSGGSGNSSGSG GSPVPSTPPTPSPST PPTPSPSASV (SEQ ID NO: 82) Long hinge 2AGSGGSGGSGGSPVP STPPTNSSSTPPTPS PSPVPSTPPTNSS STPPTPSPSPVPSTPPTNSSSTPPTPSPSA SV (SEQ ID NO: 83) 2X short hinge WSGPSGGPSSPTTGPTSTSGPEDQPLTPTG SDPQSGLGRH VVSGPSGGPSSPTTG PTSTSGPEDQPLTPTGSDPQSGLGRHLGV (SEQ ID NO: 84) Hinge TTGPTSTSGPEDQPL (truncated)TPTGSDPQSGLGRHL GV (SEQ ID NO: 93) Hinge- YGSQSSKPYLLTHPS transmembraneDPLEL VVSGPSGGPS SPTTGPTSTSGPEDQ PLTPTGSDPQSGLGR HLGVVIGILVAVILLLLLLLLLFLIL SEQ ID NO: 20 Transmembrane- VVIGILVAVILLLLL intracellularLLLLFLILRHRRQGK domain HWTSTQRKADFQHPA GAVGPEPTDRGLQWR SSPAADAQEENLYAAVKHTQPEDGVEMDTRS PHDEDPQAVTYAEVK HSRPRREMASPPSPL SGEFLDTKDRQAEEDRQMDTEAAASEAPQD VTYAQLHSLTLRREA TEPPPSQEGPSPAVP SIYATLAIH SEQ ID NO: 21Polypeptide Sequences for Illustrative Chimeric Antigen Receptors NameSequence C563 (SEQ ID NO: 47) QVQLQESGPGLVKPS DTLSLTCAVSGYSISSSNWWGWIRQPPGKG LEWIGYIYYSGSTYY NPSLKSRVTMSVDTS KNQFSLKLSSVTAVDTAVYYCARIPFGDWW YFDLWGRGTLVTVSS GGGGSGGGGSGGGGS GGDIQMTQSPSSLSASVGDRVTITCRASQS ISSYLNWYQQKPGKA PKLLIYAASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSFVLTFGGGTK VEIKTTTPAPRPPIP APTIASQPLSLRPEACRPAAGGAVHTRGLD FACDFWVLVVVGGVL ACYSLLVTVAFIIFW VRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRSKRG RKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELRVKFSR SADAPAYKQGQNQLY NELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGER RRGKGHDGL YQGLSTATKDTYDAL FIMQALPPRCl759 (SEQ ID NO: 48) DIQMTQSPSSLSASV GDRVTITCRASQSIS SYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ SYSTPLTFGGGTKVEIKGGGGSGGGGSGGG GSGGEVQLVESGGGL VQPGGSLRLSCAASG FTVYDYMSWVRQAPGKGLEWVSVIYSGGST YYADSVKGRFTISRD NSKNTLYLQMNSLRA EDTAVYYCARYSYYYYYMDVWGKGTTVTVS SFGSFRALPCWSNSS DPLLVSVTGNPSSSW PSPTEPSSKSGICRHLHVLIGTSVVIF LFILLLFFLLYRWCS NKKNAAVMDQEPAGD RTVNRQDSDEQDPQEVTYAQLDFICVFIQR KISRPSQRPKTPLTD TSVYTELPNAEPRSK WSCPRAPQSGLEGVFCl760 (SEQ ID NO: 49) DIQMTQSPSSLSASV GDRVTITCRASQSIS SYLNWYQQKPGKAPKUJYAASSLQSGVPSR FSGSGSGTDFTLTIS SLQPEDFATYYCQQS YSTPLTFGGGTKVEIKGGGGSGGGGSGGGG SGGEVQLVESGGGLV QPGGSLRLSCAASGF TVYDYMSWVRQAPGKGLEWVSVIYSGGSTY YADSVKGRFTISRDN SKNTLYLQMNSLRAE DTAVYYCARYSYYTYYMDVWGKGTTVTVSS FGSFRALPHAWSDPS DPLPVSVTGNSRNLH VLIGTSVVIIPFAILLFFLLHRWCA NKKNAVVMDQEPAGN RTVNREDSDEQDPQE VTYAQLNHCVFTQRKITRPSQRPKTPPTDT SV C1761 (SEQ ID NO: 50) DIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPK LLIYAASSLQSGVPS RFSGSGSGTDFTLTI SSLQPEDF ATYYCQQSYSTPLTFGGGTKVEIKGGGGSG GGGSGGGGSGGEVQL VESGGGLVQPGGSLR LSCAASGFTVYDYMSWVRQAPGKGLEWVSV IYSGGSTYYADSVKG RFTISRDNSKNTLYL QMNSLRAEDTAVYYCARYSYYYYYMDVWGK GTTVTVSSYGSQSSK PYLLTHPSDPIFIVV SGPSGGPSSPTTGPTSTSGPEDQPLTPTGS DPQSGLGRHLGVVIG ILVAVILLLLLLLLL FLILRHRRQGKHWTSTQRKADFQHPAGAVG PEPTDRGLQWRSSPA ADAQEENLYAAVKHT QPEDGVEMDTRSPHDEDPQAVTYAEVKHSR PRREMASPPSPLSGE FLDTKDRQAEEDRQM DTEAAASEAPQDVTYAQLHSLTLRREATEP PPSQEGPSPAVPSIY ATLAIIT C1762 (SEQ ID NO: 51)DIQMTQSPSSLSASV GDRVTITCRASQSIS SYENWYQQKPGKAPK LLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQ SYSTPLTFGGGTKVE IKGGGGSGGGGSGGGGSGGEVQLVESGGGL VQPGGSERESCAASG FTVYDYMSWVRQAPG KGLEWVSVIYSGGSTYYADSVKGRFTISRD NSKNTLYLQMNSLRA EDTAVYYCARYSYYY YYMDVWGKGTTVTVSSTERRAEVPTAHPSP SPRPAGQFQTLWGWG GLLGSLVLLVWVLAV ICSRAARGTIGARRTGQPLKEDPSAVPVFS VDYGELDFQWREKTP EPPVPCVPEQTEYAT IVFPSGMGTSSPARRGSADGPRSAQPLRPE DGHCSWPL C2057 (SEQ ID NO: 52) METLLGELILWLQLQWVSSKQEVTQIPAAL SVPEGENLVLNCSFT DSAIYNLQWFRQDPG KGLTSLLLIQSSQREQTSGRLNASLDKSSG RSTLYIAASQPGDSA TYLCAVRPLYGGSYI PTFGRGTSLFVHPYIQNPDPAVYQLRDSKS SDKSVCLFTDFDSQT NVSQSKDSDVYITDK CVLDMRSMDFKSNSAVAWSNKSDFACANAF NNSIIPEDTFFPSPE SSCDVKLVEKSFETD TNLNFQNLSVVIGILVAVILLLLLLLLLFL ILRHRRQGKFIWTST QRKADFQHPAGAVGP EPTDRGLQWRSSPAADAQEENLYAAVKIIT QPEDGVEMDTRSPHD EDPQAVTYAEVKHSR PRREMASPPSPLSGEFLDTKDRQAEEDRQM DTEAAASEAPQDVTY AQLHSLILRREATEP PPSQEGPSPAVPSIY ATLAIHC2058 (SEQ ID NO: 53) MSIGLLCCAALSLLW AGPVNAGVTQTPKFQ VLKTGQSMTLQCAQDMNHEYMSWYRQDPGM GLRLIHYSVGAGITD QGEVPNGYNVSRSTT EDFPLRLLSAAPSQTSVYFCASSYVGNTGE LFFGEGSRLTVLEDL KNVFPPEVAVFEPSE AEISHTQKATLVCLATGFYPDHVELSWWVT JGKEVHSGVCTDPQP LKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSEND EWTQDRAKPVTQIVS AEAWGRADCGFTSES YQQGVLSVVIGILVAVTLLLLLLLLLFLIL RHRRQGKHWTSTQRK ADFQHPAGAVGPEPT DRGLQWRSSPAADAQEENLYAAVKHTQPED GVEMDTRSPHDEDPQ AVTYAEVKHSRPRRE MASPPSPLSGEFLDTKDRQAEEDRQMDTEA AASEAPQDVTYAQLH SLTLRREATEPPPSQ EGPSPAVPSIYATLA IHC2070 (SEQ ID NO: 54) MSIGLLCCAALSLLW AGPVNAGVTQTPKFQ VLKTGQSMTLQCAQDMNHEYMSWYRQDPGM GLRLIHYSVGAGITD QG ATYYCQQSYSTPLTF GGGTKVEIKGGGGSGGGGSGGGGSGGEVQL VESGGGLVQPGGSLR LSCAASGFTVYDYMS YVVRQAPGKGLEWVSVIYSGGSTYYADSVK GRFTISRDNSKNTLY LQMNSLRAEDTAVYY CARYSYYYYYMDVWGKGTTVTVSSYGSQSS KPYLLTHPSDPIFIW SGPSGGPSSPTTGPT STSGPEDQPLTPTGSDPQSGLGRHLGVVIG ILVAVILLLLLLLLL FLILRHRRQGKHWTS TQRKADFQHPAGAVGPEPTDRGLQWRSSPA ADAQEENLYAAVKIT FQPEDGVEMDTRSPH DEDPQAVTYAEVKHSRPRREMASPPSPLSG EFLDTKDRQAEEDRQ MDTEAAASEAPODVT YAQLHSLTLRREATEPPPSQEGPSPAVPSI YATLAIIT C1762 (SEQ ID NO: 51) DIQMTQSPSSLSASVGDRVTITCRASQSIS SYLNWYQQKPGKAPK LLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQ SYSTPLTFGGGTKVE IKGGGGSGGGGSGGG GSGGEVQLVESGGGLVQPGGSLRESCAASG FTVYDYMSWVRQAPG KGLEWVSVIYSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARYSYYY YYMDVWGKGTTVTVS STERRAEVPTAHPSPSPRPAGQFQTLWGWG GLLGSLVLLVWVLAV ICSRAARGTIGARRT GQPLKEDPSAVPVFSVDYGELDFQWREKTP EPPVPCVPEQTEYAT IVFPSGMGTSSPARR GSADGPRSAQPLRPE DGHCSWPLC2057 (SEQ ID NO 52) METLLGLLILWLQLQ WVSSKQEVTQIPAAL SVPEGENLVLNCSFTDSAIYNLQWFRQDPG KGLTSLLLIQSSQRE QTSGRLNASLDKSSG RSTLYIAASQPGDSATYLCAVRPLYGGSYI PTFGRGTSLFVHPYI QNPDPAVYQLRDSKS SDKSVCLFTDFDSQTNVSQSKDSDVYITDK CVLDMRSMDFKSNSA VAWSNKSDFACANAF NNSHPEDTFFPSPESSCDVKLVEKSFETDT NLNFQNLSVVIGILV AVILLLLLLLLLFLI LRHRRQGKFIWTSTQRKADFQHPAGAVGPE PTDRGLQWRSSPAAD AQEENLYAAVKIITQ PEDGVEMDTRSPHDEDPQAVTYAEVKHSRP RREMASPPSPLSGEF LDTKDRQAEEDRQMD TEAAASEAPQDVTYAQLHSLTLRREATEPP PSQEGPSPAVPSIYA TLAIH C2058 (SEQ ID NO: 53)MSIGLLCCAALSLLW AGPVNAGVTQTPKFQ VLKTGQSMTLQCAQD MNHEYMSWYRQDPGMGLRLIHYSVGAGITD QGEVPNGYNVSRSTT EDFPLRLLSAAPSQT SVYFCASSYVGNTGELFFGEGSRLTVLEDL KNVFPPEVAVFEPSE AEISHTQKATLVCLA TGFYTPHVELSWWVNGKEVHSGVCTDPQPL KEQPALNDSRYCLSS RLRVSATFWQNPRNH FRCQVQFYGLSENDEWTQDRAKPVTQIVSA EAWGRADCGFTSESY QQGVLSVVIGILVAV ILLLLLLLLLFLILRHRRQGKHWTSTQRKA DFQHPAGAVGPEPTD RGLQWRSSPAADAQE ENLYAAVKHTQPEDGVEMDTRSPHDEDPQA VTYAEVKHSRPRREM ASPPSPLSGEFLDTK DRQAEEDRQMDTEAAASEAPQDVTYAQLHS LTLRREATEPPPSQE GPSPAVPSIYATLAI H C2070 (SEQ ID NO: 54)MSIGLLCCAALSLLW AGPVNAGVTQTPKFQ VLKTGQSMTLQCAQD MNHEYMSWYRQDPGMGLRLIHYSVGAGITD QGEVPNGYNVSRSTT EDFPLRLLSAAPSQT SVYFCASSYVGNTGELFFGEGSRLTVLEDL KNVFPPEVAVFEPSE AEISHTQKATLVCLA TGFYPDHVELSWWVNGKEVHSGVCTDPQPL KEQPALNDSRYCLSS RLRVSATFWQNPRNH FRCQVQFYGLSENDEWTQDRAKPVTQIVSA EAWGRADGGFTSESY QQGVLSATILYEILL GKATLYAVLVSALVLMAMVKRKDSRGGGGG SGGGGSGGGGSRAAR GTIGARRTGQPLKED PSAVPVFSVDYGELDFQWREKTPEPPVPCV PEQTEYATIVFPSGM GTSSPARRGSADGPR SAQPLRPEDGHCSWP LC2071 (SEQ ID NO: 55) MSIGLLCCAALSLLW AGPVNAGVTQTPKFQ VLKTGQSMTLQCAQDMNHEYMSWYRQDPGM GLRLIHYSVGAGITD QGIEVPNGYNVSRST TEDFPLRLLSAAPSQTSVYFCASSYVGNTG ELFFGEGSRLTVLED LKNVFPPEVAVFEPS EAEISHTQKATLVCLATGFYPDHVELSWWV NGKEVHSGVCTDPQP LKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSEND EWTQDRAKPVTQIVS AEAWGRADCGFTSES YQQGVLSATILYEILLGKATLYAVLVSALV LMAMVKRKDSRGGGG GSGGGGSGGGGSNKK NAAVMDQEPAGDRTVNRQDSDEQDPQEVTY AQLDHCVFIQRKISR PSQRPKTPLTDTSVY TELPNAEPRSKVVSCPRAPQSGLEGVF C2072 (SEQ ID NO: 56) MSIGLLCCAALSLLW AGPVNAGVTQTPKFQVLKTGQSMTLQCAQD MNHEYMSWYRQDPGM GLRLIHYSVGAGITD QGEVPNGYNVSRSTTEDFPLRLLSAAPSQT SVYFCASSYVGNTGE LFFGEGSRLTVLEDL KNVFPPEVAVFEPSEAEISHTQKATLVCLA TGFYPDHVELSWWVN GKEVHSGVCTDPOPL KEQPALNDSRYCLSSRLRVSATFWQNPRNH FRCQVQFYGLSENDE WTQDRAKPVTQIVSA EAWGRADCGFTSESYQQGVLSATILYEILL GKATLYAVLVSALVL MAMVKRKDSRGGGGG SGGGGSGGGGSRHRRQGKHWTSTQRKADFQ HPAGAVGPEPTDRGL QWRSSPAADAQEENL YAAVKHTQPEDGYEMDTRSPHDEDPQAVTY AEVKHSRPRREMASP PSPLSGEFLDTKDRQ AEEDRQMDTEAAASEAPQDVTYAQLHSLTL RREATEPPPSQEGPS PAVPSIYATLAIH C2106 (SEQ ID NO: 57)DIQMIQSPSSLSASV GDRVIITCRASQSIS SYLNWYQQKPGKAPK LLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQ SYSTPLTFGGGTKVE IKGGGGSGGGGSGGGGSGGEVQLVESGGGL VQPGGSLRLSCAASG FTVYDYMSWVRQAPG KGLEWVSVIYSGGSTYYADSVKGRFTISRD NSKNTLYLQMNS LRAEDTAVYYCARYS YYYYYMDVWGKGTTVTVSSTTTPAPRPPTP APTIASQPLSLRPEA CRPAAGGAVHTRGLD FACDIYIYVAPLAGTCGVLLLSLVITLYCN HDREKKPRQHSGDHE NEMNVPSDKEMFSRS VTSLATDAPASSEQNGALTNGDILSEDSTL TCMQHYEEVQTSASD LLDSQDSTGKPKCFI QSRELPRIPPESAVDTMLTARSVDGDQGLG MEGPYEVLKDSSSQE NMVEDCLYETVKEIK EVAAAAHLEKGHSGKAKSTSASKELPGPQT EGKAEFAEYASVDRN KKCRQSVNVESI LGNSCDPEEEAPPPVPVKLLDENENLQEKE GGEAEESATDTTSE TNKRFSSLSYKSR EEDPTLTEEEISAMYSSVNKPGOLVNKSGO SLTVPESTYTSIQGD PQRSPSSCNDLYATV KDFEKTPNSTLPPAGRPSEEPEPDYEAIQT LNREEEKATLGTNGH HGLVPKENDYESISD LQQGRDITRLC2107 (SEQ ID NO: 58) DIQMTQSPSSLSASV GDRVTITCRASQSIS SYLNWYQQKPGKAPKLLIYAA SSLQSGVPSRFSGSG SGTDFTLTISSLQPE DFATYYCQQSYSTPLTFGGGTKVEIKGGGG SGGGGSGGGGSGGEV QLVESGGGLVQPGGS LRLSCAASGFTVYDYMSWVRQAPGKGLEWV SVIYSGGSTYYADSV KGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCARYSYYYYYMDVW GKGTTVTVSSTTTPA PRPPTPAPTIASQPL SLRPEACRPAAGGAVHTRGLDFACDLWGSL AAVAIFFVITFLIFL CSSCDREKKPRQHSG DHENLMNVPSDKEMFSRSVTSLATDAPASS EQNGALTNGDILSED STLTCMQHYEEVQTS ASDLLDSQDSTGKPKCHQSRELPRIPPESA VDTMLTARSVDGDQG LGMEGPYEVLKDSSS QENMVEDCLYETVKEIKEVAAAAHLEKGHS GKAKSTSASKELPGP QTEGKAEFAEYASVD RNKKCRQSVNVESILGNSCDPEEEAPPPVP VKLLDENENLQEKEG GEAEESATDTTSETN KRFSSLSYKSREEDPTLTEEEISAMYSSVN KPGQLVNKSGQSLTV PESTYTSIQGDPQRS PSSCNDLYATVKDFEKTPNSTLPPAGRPSE EPEPDYEAIQTLNRE EEKATLGTNGHHGLV PKENDYESISDLQQG RDITRLC2153 (SEQ ID NO: 59) DIQMTQSPSSLSASV GDRVTITCRASQSI SSYLNWYQQKPGKAPKLLIYAA SSLQSGVPSRFSGSG SGTDFTLTISSLQPE DFATYYCQQSYSTPLTFGGGTKVEIKGGGG SGGGGSGGGGSGGEV QLVESGGGLVQPGGS LRLSCAASGFTVYDYMSWVRQAPGKGLEWV SVIYSGGSTYYADSV KGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCARYSYYYYYMDVW GKGTTVTVSSTTTPA PRPPTPAPTIASQPL SLRPEACRPAAGGAVHTRGLDFACDFWVLW VGGVLACYSLLVTVA FIIFWVLRHRRQGKH WTSTQRKADFQHPAGAVGPEPTDRGLQWRS SPAADAQEENLYAAV KHTQPEDGVEMDTRS PHDEDPQAVTYAEVKHSRPRREMASPPSPL SGEFLDTKDRQAEED RQMDTEAAASEAPQD VTYAQLHSLTLRREATEPPPSQEGPSPAVP SIYATLAIH C2156 (SEQ ID NO:60) METLLGLLILWLQLQWVSSKQEVTQIPAAL SVPEGENLVLNCSFT DSAIYNLQWFRQDPG KGLTSLLLIQSSQREQTSGRLNASLDKSSG RSTLYIAASQPGDSA TYLCAVRPLYGGSYI PTFGRGTSLIVHPYIQNPDPAVYQLRDSKS SDKSVCLFTDFDSQT NVSQSKDSDVYITDK CVLDMRSMDFKSNSAVAWSNKSDFACANAF NNSIIPEDTFFPSPE SSCDVKLVEKSFETD TNLNFQNLSVIGFLILLLLVAGFNLLMTLL LWSSLILRHRRQGKH WTSTQRKADFQHPAG AVGPEPTDRGLQWRSSPAADAQEENLYAAV KHTQPEDGVEMDTRS PHDEDPQAVTYAEVK HS RPRREMASPPSPLSGEFLDTKDRQAEEDRQ MDTEAAASEAPQDVT YAQLHSLTLRREATE PPPSQEGPSPAVPSI YATLAIHC2157 (SEQ ID NO: 61) MSIGLLCCAALSLLW AGPVNAGVTQTPKFQ VLKTGQSMTLQCAQDMNHEYMSWYRQDPGM GLRLIHYSVGAGITD QGEVPNGYNVSRSTT EDFPLRLLSAAPSQTSVYFCASSYVGNTGE LFFGEGSRLTVLEDL KNWPPEVAVFEPSEA EISHTQKATLVCLATGFYPDIIVELSWWVN GKEVHSGVCTDPQPL KEQPALNDSRYCLSS RLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSA EAWGRADCGFTSESY QQGVLSATILYLILLGLATLYAVLVSALVL MLILRIIRRQGKHWT STQRKADFQHPAGAV GPEPTDRGLQWRSSPAADAQEENLYAAVKH TQPEDGVEMDTRSPH DEDPQAVTYAEVKHS RPRREMASPPSPLSGEFLDTKDRQAEEDRQ MDTEAAASEAPQDVT YAQLHSLTLRREATE PPPSQEGPSPAVPSI YATLAIHC2158 (SEQ ID NO: 62) QVQLQESGPGLVKPS DTLSLTCAVSGYSIS SSNWWGWIRQPPGKGLEWIGYIYYSGSTYY NPSLKSRVTMSVDTS KNQFSLKLSSVTAVD TAVYYCARIPFGDWWYFDLWGRGTLVTVSS GGGGSGGGGSGGGGS GGDIQMTQSPSSLSA SVGDRVTITCRASQSISSYLNWYQQKPGKA PKLLIYAASSLQSGV PSRFSGSGSGTDFTL TISSLQPEDFATYYCQQSYSFVLTFGGGTK VEIKTTTPAPRPPTP APTIASQPLSLRPEA CRPAAGGAVHTRGLDFACDFWVLVVVGGVL ACYSLLVTVAFIIFV VVLRHRRQGKHWTST QRKADFQHPAGAVGPEPTDRGLQWRSSPAA DAQEENLYAAVKHTQ PEDGVEMDTRSPHDE DPQAVTYAEVKHSRPRREMASPPSPLSGEF LDTKDRQAEEDRQMD TEAAASEAPQDVTYA QLHSLTLRREATEPPPSQEGPSPAVPSIYA TLAIH C2179 (SEQ ID NO: 63) DIQMTQSPSSLSASVGDRVTITCRASQSIS SYLNWYQQKPGKAPK LLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQ SYSTPLTFGGGTKVE IKGGGGSGGGGSGGG GSGGEVQLVESGGGLVQPGGSLRLSCAASG FTVYDYMSWVRQAPG KGLEWVSVIYSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARYSYYY YYMDVWGKGTTVTVS SYGSQSSKPYLLTHPSDPLELVVSGPSGGP SSPTTGPTSTSGPED QPLTPTGSDPQSGLG RHLGVVIGILVAVILLLLLLLLLFLILRHR RQRPRREMASPPSPL SGEFLDTKDRQAEED RQMDTEAAASEAPQDVTYAQLHSLTLRREA TEPPPSQEGPSPAVP SIYATLAIH C2180 (SEQ ID NO: 64)DIQMTQSPSSLSASV GDRVTITCRASQSIS SYLNWYQQKPGKAPK LLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQ SYSTPLTFGGGTKVE IKGGGGSGGGGSGGGGSGGEVQLVESGGGL VQPGGSLRLSCAASG FTVYDYMSWVRQAPG KGLEWVSVIYSGGSTYYADSVKGRFTISRD NSKNTLYLQMNSLRA EDTAVYYCARYSYYY YYMDVWGKGTTVTVSSYGSQSSKPYLLTHP SDPLELVVSGPSGGP SSPTTGPTSTSGPED QPLTPTGSDPQSGLGRHLGVVIGILVAVIL LLLLLL LFLILRHRRQGKHWT STQRKADFQHPAGAV GPEPTDRGLQWRSSPAADAQEENLYAAVKH TQPEDGVEMDTRSPH DEDPQAVTYAEVKFI SRPRREMASPPSPLSGEFLDTKDRQAEEDR QMDTEAAASEAPQDV TYAQLHSLTLRREAT EPPPSQEGPSPAVPSIYATLAIHRPRREMA SPPSPLSGEFLDTKD RQAEEDRQMDTEAAA SEAPQDVTYAQLHSLTLRREATEPPPSQEG PSPAVPSIYATLAIH C2181 (SEQ ID NO: 65) DIQMTQSPSSLSASVGDRVTITCRASQSIS SYLNWYQQKPGKAPK LLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQ SYSTPLTFGGGTKVE IKGGGGSGGGGSGGG GSGGEVQLVESGGGLVQPGGSLRLSCAASG FTVYDYMSWVRQAPG KGLEWVSVIYSGGST YYADSVKGRITISRDNSKNTLYLQMNSLRA EDTAVYYCARYSYYY YYMDVWGKGTTVTVS SYGSQSSKPYLLTHPSDPLELWSGPSGGPS SPTTGPTSTSGPEDQ PLTPTGSDPQSGLGR HLGVVIGILVAVILLLLLLLLLFLILRHRR QRPRREMASPPSPLS GEFLDTKDRQAEEDR QMDTEAAASEAPQDVTYAQLHSLTLRREAT EPPPSQEGPSPAVPS IYATLAIHRPRREMA SPPSPLSGEFLDTKDRQAEEDRQMDTEAAA SEAPQDVTYAQLHSL TLRREATEPPPSQEG PSPAVPSIYATLAIHC2182 (SEQ ID NO: 66) DIQMTQSPSSLSASV GDRVTITCRA SQSISSYLNWYQQKPGKAPKLLIYAA SSLQSGVPSRFSGSG SGTDFTLTISSLQPE DFATYYCQQSYSTPLTFGGGTKVEIKGGGG SGGGGSGGGGSGGEV QLVESGGGLVQPGGS LRLSCAASGFTVYDYMSWVRQAPGKGLEWV SVIYSGGSTYYADSV KGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCARYSYYYYYMDVW GKGTTVTVSSYGSQS SKPYLLTHPSDPLEL VVSGPSGGPSSPTTGPTSTSGPEDQPLTPT GSDPQSGLGRHLGVV IGILVAVILLLLLLL LLFLILRHRRQGKHWTSTQRKADFQHPAGA VGPEPTDRGLQWRSS PAADAQEENLFAAVK HTQPEDGVEMDTRSPHDEDPQAVTFAEVKH SRPRREMASPPSPLS GEFLDTKDRQAEEDR QMDIEAAASEAPQDVTFAQLHSLTLRREAT EPPPSQEGPSPAVPS IFATLAIH C2183 (SEQ ID NO. 67)DIQMTQSPSSLSASV GDRVTITCRASQSIS SYLNWYQQKPGKAPK LLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQ SYSTPLTFGGGTKVE IKGGGGSGGGGSGGGGSGGEVQLVESGGGL VQPGGSLRLSCAASG FTVYDYMSWVRQAPG KGLEWVSVIYSGGSTYYADSVKGRFTISRD NSKNTLYLQMNSLRA EDTAVYYCARYSYYY YYMDVWGKGTTVTVSSYGSQSSKPYLLTHP SDPLELWSGPSGGPS SPTTGPTSTSGPEDQ PLTPTGSDPQSGLGRHLGVVIGILVAVILL LLLLLLLFLILRHRR QGKHWTSTQRKADFQ HPAGAVGPEPTDRGLQWRSSPAADAQEENL FAAVKHTQPEDGVEM DTRSPHDEDPQAVTF AEVKHSRPRREMASPPSPLSGEFLDTKDRQ AEEDRQMDTEAAASE APQDVTYAQLHSLTL RREATEPPPSQEGPSPAVPSIYATLAIH C2184 (SEQ ID NO: 68) DIQMTQSPSSLSASV GDRVTITCRASQSISSYLNWYQQKPGKAPK LLIYAASSLQSGVPS RFSGSGSGTDFTLTI SSLQPEDFATYYCQQSYSTPLTFGGGTKVE IKGGGGSGGGGSGGG GSGGEVQLVESGGGL VQPGGSLRLSCAASGFTVYDYMSWVRQAPG KGLEWVSVIYSGGST YYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAVYYCARYSYYY YYMDVWGKGTTVTVS SYGSQSSKPYLLTHP SDPLELVVSGPSGGPSSPTTGPTSTSGPED QPLTPTGSDPQSGLG RHLGVVIGILVAVIL LLLLLLLLFLILRHRRQGKHWTSTQRKADF QHPAGAVGPEPTDRG LQWRSSPAADAQEEN LYAAVKHTQPEDGVEMDTRSPHDEDPQAVT YAEVKFISRPRREMA SPPSPLSGEFLDTKD RQAEEDRQMDTEAAASEAPQDVTFAQLHSL TLRREATEPPPSQEG PSPAVPSIFATLAIH C2218 (SEQ ID NO: 69)DIQMTQSPSSLSASV GDRVTITCRASQSIS SYLNWYQQKPGKAPK LLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQ SYSTPLTFGGGTKVE IKGGGGSGGGGSGGGGSGGEVQLVESGGGL VQPGGSLRLSCAASG FTVYDYMSWVRQAPG KGLEWVSVIYSGGSTYYADSVKGRFTISR DNSKNTLYLQMNSLR AEDTAVYYCARYSYY YYYMDVWGKGTTVTVSSYGSQSSKPYLLTH PSDPLELVVSGPSGG PSSPTTGPTSTSGPE DQPLTPTGSDPQSGLGRHLGVVIGILVAVI LLLLLLLLLFLILRR HQGKQNELSDTAGRE INLVDAHLKSEQTEASTRQNSQVLLSETGI YDNDPDLCFRMQEGS EVYSNPCLEENKPGI VYASLNHSVIGPNSRLARNYTCEAPTEYAS ICVRS C2219 (SEQ ID NO: 70) DIQMTQSPSSLSASVGDRVTITCRASQSIS SYLNWYQQKPGKAPK LLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQ SYSTPLTFGGGTKVE IKGGGGSGGGGSGGG GSGGEVQLVESGGGLVQPGGSLRLSCAASG FTVYDYMSWVRQAPG KGLEWVSVIYSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARYSYYY YYMDVWGKGTTVTVS SYGSQSSKPYLLTHPSDPLELVVSGPSGGP SSPTTGPTSTSGPED QPLTPTGSDPQSGLG RHLGVLLPLGGLPLLITTCFCLFCCLRRHQ GKQNELSDTAGREIN LVDAHLKSEQTEAST RQNSQVLLSETGIYDNDPDLCFRMQEGSEV YSNPCLEENKPGIVY ASLNHSVIGPNSRLA RNVKEAPTEYASICV RSC2220 (SEQ ID NO: 71) DIQMTQSPSSLSASV GDRVTITCRASQSIS SYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ SYSTPLTFGGGTKVEIKGGGGSGGGGSGGG GSGGEVQLVESGGGL VQPGGSLRLSCAASG FTVYDYMSWVRQAPGKGLEWVSVIYSGGST YYADSVKGRFTISRD NSKNTLYLQMNSLRA EDTAVYYCARYSYYYYYMDVWGKGTTVTVS STDVKSASERPSKDE MASRPWLLYRLLPLG GLPLLITTCFCLFCCLRRHQGKQNELSDTA GREINLVDAHLKSEQ TEASTRQNSQVLLSE TGIYDNDPDLCFRMQEGSEVYSNPCLEENK PGIVYASLNHSVIGP NSRLARNVKEAPTEY ASICVRSC2302 (SEQ ID NO: 77) DIQMTQSPSSLSASV GDRVTITCRASQSIS SYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTI SSLQPEDFATYYCQQ SYSTPLTFGGGTKVEIKGGGGSGGGGSGGG GSGGEVQLVESGGGL VQPGGSLRLSCAASG FTVYDYMSWVRQAPGKGLEWVSVIYSGGST YYADSVKGRFTISRD NSKNTLYLQMNSLRA EDTAVYYCARYSYYYYYMDVWGKGTTVTVS SYGSQSSKPYLLTHP SDPLELVVSGPSGGP SSPTTGPTSTSGPEDQPLTPTGSDPQSGLG RHLGVVIGILVAVIL LLLLLLLLFLILRHR RQRPRREMASPPSPLSGEFLDTKDRQAEED RQMDTEAAASEAPQD VTYAQLHSLTLRREA TEPPPSQEGPSPAVPSIYATLAIIIRPRRE MASPPSPLSGEFLDT KDRQAEEDRQMDTEA AASEAPQDVTYAQLHSLTLRREATEPPPSQ EGPSPAVPSIYAPLA OIRPRREMASPPSPL SGEFLDTKDRQAEEDRQMDTEAAASEAPQD VTYAQLMSLTLRREA TEPPPSQEGPSPAVP SIYATLAIHCT138 (SEQ ID NO: 78) MGPVTCSVLVLLLML RRSNGDGDSVTQTEG LVTLTEGLPVMLNCTYQTIYSNPFLFWYVQ HLNESPRLLLKSFTD NKRTEHQGFHATLHK SSSSFHLQKSSAQLSDSALYYCAFDTNTYK VIFGKGTHLHVLPNI QNPEPAVYQLKDPRS QDSTLCLFTDFDSQINVPKTMESGTFITDK TVLDMKAMDSKSNGA IAWSNQTSFTCQDIF KETNTTYPSSDVPCDATLTEKSFETDMNLN FQNLSVMGLRILLLK VAGFNLLMTLRLWSS RAKRSGSGATNFSLLKQAGDVEENPGPMRV RLISAVVLCSLGTGL VDMKVTQMPRYLIKR MGENYTLECGQDMSHETMYWYRQDPGLGLQ LIYISYDVDSNSEGD IPKGYRVSRKKREHF SLILDSAKTNQTSVYFCASSSTNTEVFFGK GTRLTWEDLRNVTPP KVSLFEPSKAEIANK QKATLVCLARGFFPDHVELSWWVNGKEVHS GVSTDPQAYKESNYS YCLSSRLRVSATFWH NPRNHFRCQVQFHGLSEEDKWPEGSPKPVT QNISAEAWGRADCGI TSASYQQGVLSATIL YEILLGKATLYAVLVSTLVVMAMVKRKNS CT139 (SEQ ID NO: 79) MVLVTILLLSAFFSL RGNSAQSVDQPDAFIVTLSEGASLELRCSY SYSAAPYLFVVYVQY PGQSLQFLLKYITGD TVVKGTKGFEAEFRKSNSSFNLKKSPAHWS DSAKYFCALEGPDTG NYKYVFGAGTRLKVI AHIQNPEPAVYQLKDPRSQDSTLCLFTDFD SQINWKTMESGTFIT DKTVLDMKAMDSKSN GAIAWSNQTSFTCQDIFKETNATYPSSDVP GDATLTEKSFETDMN LNFQNLSVMGLRILL LKVAGFNLLMTLRLWSSRAKRSGSGATNFS LLKQAGDVEENPGPM GIQTLCCVLFYVLIA NHTDAGVTQTPRHEVAEKGQTIILKCEPVS GHNDLFWYRQTKIQG LELLSYFRSKSLMED GGAFKDRFKAEMLNSSFSTLKIQPTEPRDS AVYLCASSFGTASAE TLYFGSGTRLTVLED LRNVTPPKVSLFEPSKAEIANKQKATLVCL ARGFFPDHVELSWWV NGKEVHSGVSTDPQA YKESNYSYCLSSRLRVSATFWHNPRNHFRC QVQFHGLSEEDKWPE GSPKPVTQNISAEAW GRADC GITSASYHQGVLSATILYEILLGKATLYAV LVSGLVLMAMVKKKN S Cl765 with MDMRVPAQLLGLLLLsignal peptide WLRGARCDVLMTQTP (SEQ ID NO: 120), LSLPVSLGDQASISC LIR1RSSQSIVHSNGNTYL hinge, TM and EWYLQKPGQSPKLLI ICD are YKVSNRFSGVPDRFSunderlined GSGSGTDFTLKISRV Polynucleotide EAEDLGVYYCFQGSH sequence-VPRTSGGGTKLEIKG SEQ ID NO: 121 GGGSGGGGSGGGGSG GQVQLQQSGPELVKPGASVRISCKASGYTF TSYHIHWVKQRPGQG LEWIGWIYPGNVNTE YNEKFKGKATLTADKSSSTAYMHLSSLTSE DSAVYFCAREEITYA MDYWGQGTSVTVSSY GSQSSKPYLLTHPSDPLELVVSGPSGGPSS PTTGPTSTSGPEDQP LTPTGSDPQSGLGRH LGVVIGILVAVILLLLLLLLLFLELRHRRQ GKHWTSTQRKADFQH PAGAVGPEPTDRGLQ WRSSPAADAQEENLYAAVKHTQPEDGVEMD TRSPHDEDPQAVTYA EVKHSRPRREMASPP SPLSGEFLDTKDRQAEEDRQMDTEAAASEA PQDVTYAQLHSLTLR REATEPPPSQEGPSP AVPSIYATLAIHCl765 without DVLMTQTPLSLPVSL signal GDQASISCRSSQSIV peptideHSNGNTYLEWYLQKP (SEQ ID NO: 122), GQSPKLLIYKVSNRF LD11 hinge,SGWDRFSGSGSGTDF TM and TLKISRVEAEDLGVY ICD are YCFQGSHVPRTSGGGunderlined TKLEIKGGGGSGGGG Polynucleotide SGGGGSGGQVQLQQS sequence-GPELVKPGASVRISC SEQ ID NO: 123 KASGYTFTSYHIHWV KQRPGQGLEWIGWIYPGNVNTEYNEKFKGK ATLTADKSSSTAYMH LSSLTSEDSAVYFCA REEITYAMDYWGQGTSVTVSSYGSQSSKPY LLTHPSDPLELVVSG PSGGPSSPTTGPTST SGPEDQPLTPTGSDPQSGLGRHLGVVIGIL VAVILLLLLLLLLFL ILRHRRQGKHWTSTQ RKADFQHPAGAVGPEPTDRGLQWRSSPAAD AQEENLYAAVKHTQP EDGVEMDTRSPHDED PQAVTYAEVKHSRPRREMASPPSPLSGEFL DTKDRQAEEDRQMDT EAAASEAPQDVTYAQ LHSLTLRREATEPPPSQEGPSPAPSIYAIL AIH

The present description sets forth numerous exemplary configurations,methods, parameters, and the like. It should be recognized, however,that such description is not intended as a limitation on the scope ofthe present disclosure, but is instead provided as a description ofexemplary embodiments.

EXAMPLES Example 1: LILRB1-Based Inhibitory scFv-CAR Compared to PD-1,EJR3DL2, EJR3DL3

The NY-ESO-1-responsive inhibitory construct was created by fusing theNY-ESO-1 ligand binding scFv domain (C-266) to domains of receptorsincluding hinge, transmembrane region, and/or intracellular domain ofLeukocyte immunoglobulin-like receptor subfamily B member 1, LILRB1(LILRB1); Killer cell immunoglobulin-like receptor 3DL12, KIR3DL2;Killer cell immunoglobulin-like receptor 3DL3, KIR3DL3; and/or B- andT-lymphocyte attenuator, BTLA. Gene segments were combined using GoldenGate cloning and inserted downstream of an eF1α promoter contained in alentiviral expression plasmid (pLenti1).

As reporter cells, Jurkat cells encoding an NFAT Luciferase reporterwere maintained in RPMI media supplemented with 10% FBS, 1% Pen/Strepand 0.4 mg/mL G418/Geneticin. T2 cells (ATCC CLR-1992) were maintainedin IMDM media+20% FBS and 1% Pen/Strep. For each construct to beevaluated, Jurkat cells were transfected via 100 μL format Neonelectroporation system (Thermo Fisher) according to manufacturer'sprotocol using the following settings: 3 pulses, 1500V, 10 msec.

Co-transfection was performed with 3 μg of activating CAR construct(C-563) or TCR construct (CT-139) and 3 μg of either inactivating CARconstruct or empty vector (pLenti0) per 1 million cells and recovered inRPMI media supplemented with 20% heat-inactivated FBS and 0.1%Pen/Strep.

Peptides, MAGE-A3 (FLWGPRALV) (SEQ ID NO: 106) and modified NY-ESO-1(SLLMWITQV) (SEQ ID NO: 107), were synthesized by Genscript. Activatingpeptide, MAGE-A3, was serially diluted 5-fold starting at 50 μM.Inactivating peptide, NY-ESO-1, was diluted to 50 μM, 5 μM, 0.5 μM, or0.05 uM and these constant amounts were added to the MAGE-A3 serialdilutions and subsequently loaded onto 10,000 T2 cells in 15 μL of RPMIsupplemented with 1% BSA and 0.1% Pen/Strep and incubated in Corning®384-well Low Flange White Flat Bottom Polystyrene TC-treatedMicroplates. The following day, 10,000 Jurkat cells were resuspended in15 uL of RPMI supplemented with 10% heat-inactivated FBS and 0.1%Pen/Strep, added to the peptide-loaded T2 cells and co-cultured for 6hours. ONE-Step Luciferase Assay System (BPS Bioscience) was used toevaluate Jurkat luminescence. Assays were performed in technicalduplicates.

LILRB1 Compared to PD-1, KIR3DL2, KIR3DL3

FIG. 4 shows testing of the constructs provided in Table 1. The datashow scFv-LILRB1 inhibits CAR activation in trans. Because the constructwith an LILRB1 domain demonstrates inhibition of signaling at higherconcentrations of MAGE-A3 activator peptide, the data demonstrate that aCAR with hinge, transmembrane domain, and intracellular domain fromLILRB1 is superior to CARs generated with the same domains from PD-1,KIR3DL2, or KIR3DL3.

TABLE 1 LBD Hinge TM ICD C563 MAGE-A3 pep1 scFv CD8[117-161]CD28[135-161] CD28[136-202] 41BB[197-238] CD3z[31-127] C1761 NY-ESO-1scFv LILRB1 LILRB1 LILRB1 C1759 NY-ESO-1 scFv KIR3DL2 KIR3DL2 KIR3DL2C1760 NY-ESO-1 scFv KIR3DL3 KIR3DL3 KIR3DL3 C1762 NY-ESO-1 scFv PD1 PD1PD1 CT139 MAGE-A3 pep1 TCR (both alpha and beta chains)

Inhibition by LILRB1-Based CAR Requires Antigen Recognition by itsLigand Binding Domain

FIG. 5 shows testing of selected constructs from Table 1. The data showthat LILRB1 inhibits signaling in trans in a dose-dependent fashion. Atincreasing concentrations of the inhibitory peptide NY-ESO-1 theresponse to activating peptide MAGE-A3 shifts downward. This shows thatthe inhibitory effect of the LILRB1-based CAR is dependent on engagementof the antigen to which the ligand binding domain is specific.

LILRB1 CAR Inhibits Signaling Through the T-Cell Receptor (ICR)

FIG. 6 shows testing of selected constructs from Table 1, in particulara TCR specific for activating peptide MAGE-A3 rather than the CARs usedin previous experiments. The LILRB1-based inhibitory CAR inhibitsTCR-mediated signaling in a dose-dependent fashion.

LIRLB1 CAR Inhibition is Preserved when Two of the Four Native ITIMs arePresent

FIG. 7 show testing of LILRB1 intracellular domains having inactivatingmutations in the ITIM motifs of LILRB1. Mutation of tyrosine tophenylalanine (Y→F) was used to inactivate the ITIMs indicated by astarred number in Table 2.

TABLE 2 LBD Hinge TM ICD C563 MAGE-A3 CD8[117-161] CD28[135-161]CD28[136-202] pep1 scFv 41BB[197-238] CD3z[31-127] C1761 NY-ESO-1 scFvLILRB1 LILRB1 LILRB1 (ITIMIs: 1, 2, 3, 4) C2182 NY-ESO-1 scFv LILRB1LILRB1 LILRB1 (ITIMs: 1*, 2*, 3*, 4*) C2183 NY-ESO-1 scFv LILRB1 LILRB1LILRB1 (ITIMs: 1*, 2*, 3, 4) C2184 NY-ESO-1 scFv LILRB1 LILRB1 LILRB1(ITIMs: 1, 2, 3*, 4*)

Inactivation of all four ITIMs resulted in a non-functional inhibitoryCAR (C2182). Inactivation of only two of the four ITIMs preserved theinhibitor function of the CAR at the concentrations tested (C1760 andC1762). Inactivation of all four ITIM (C1759) demonstrates that theITIMs are necessary for inhibitory function. When all four ITIMs aremutated, the molecule loses inhibitory function. When only two of thefour ITIMs are mutated, inhibitory activity is retained.

FIG. 8 shows testing of LILRB1 ITIMs in combinations different fromthose of the native LILRB1 intracellular domain. As shown in Table 3,CARs having the third and fourth ITIMs of LILRB1 in four or six totalcopies achieve inhibitory activity comparable to the native LILRB1intracellular domain. Inhibitory activity is also observed when only onecopy each of the third and fourth ITIMs are used (C2179) or when twocopies of each ITIM is used (C2180) or when multiple copies are used(C2302 or C2180).

TABLE 3 LBD Hinge TM ICD C563 MAGE-A3 CD8[117-161] CD28[135-161]CD28[136-202] pep1 scFv 41BB[197-238] CD3z[31-127] C1761 NY-ESO-1 scFvLILRB1 LILRB1 LILRB1 (ITIMs: 1, 2, 3, 4) C2302 NY-ESO-1 scFv LILRB1LILRB1 LILRB1 (ITIMs: 3, 4, 3, 4, 3, 4) C2181 NY-ESO-1 scFv LILRB1LILRB1 LILRB1 (ITIMs: 3, 4, 3, 4) C2180 NY-ESO-1 scFv LILRB1 LILRB1LILRB1 (ITIMs: 1, 2, 3, 4, 3, 4) C2179 NY-ESO-1 scFv LILRB1 LILRB1LILRB1 (ITIMs: 3, 4)

Example 2: LILRB1 Hinge and Transmembrane Domains Enhance the InhibitoryActivity of BTLA-Based Inhibitory CARs

BTLA-Based CAR and LILRB1/BTLA-Based CARs Inhibit Signaling Via NFAT

B- and T-lymphocyte attenuator (BTLA), also known as CD272 (cluster ofdifferentiation 272) interacts with B7 homology B7H4. Unlike CTLA-4 andPD-1, it is also a ligand for tumour necrosis factor (receptor)superfamily, member 14 (TNFRSF14). Inhibitory signaling through BTLAoccurs in response to binding of B7H4 or TNFRSF14.

The full length BTLA protein was cloned into a construct having anextracellular scFv domain (C2220), a construct replacing theextracellular domain of BTLA with that of LILRB1 (C2219), and aconstruct replacing the extracellular and transmembrane domains of BTLAwith those of LILRB1 (C2218) were generated and tested, as shown inTable 4 and FIG. 9. The LILRB1-BTLA fusion exhibits inhibitory signalingcomparable to the LILRB1-based CAR.

TABLE 4 LBD Hinge TM ICD C563 MAGE-A3 CD8[117-161] CD28[135-161]CD28[136-202] pep1 scFv 41BB[197-238] CD3z[31-127] C1761 NY-ESO-1 scFvLILRB1 LILRB1 LILRB1 C2218 NY-ESO-1 scFv LILRB1 LILRB1 BTLA C2219NY-ESO-1 scFv LILRB1 BTLA BLTA C2220 NY-ESO-1 scFv BTLA BTLA BTLA

Example 3: LILRB1 Hinge and Transmembrane Compared to CD8 or CD28

LILRB1-Based CA1 with LILRB1 Hinge and Transmembrane is Superior to CD8Hinge and CD28 Transmembrane Region

The LILRB1-based CAR was compared to a CAR having the LILRB1intracellular domain but CD8 hinge and CD28 transmembrane region, asshown in Table 5 and FIG. 10.

TABLE 5 LBD Hinge TM ICD C563 MAGE-A3 CD8[117-161] CD28[135-161]CD28[136-202] pep1 scFv 41BB[197-238] CD3z[31-127] C1761 NY-ESO-1 scFvLILRB1 LILRB1 LILRB1 C2153 NY-ESO-1 scFv CD8 C28 LILRB1

The LILRB1 hinge and transmembrane region generate results surprisinglysuperior to a construct having the CD8 hinge and CD28 transmembraneregions. Emin is decreased. Overall dynamic range is increased.Inhibitory potency is increased.

TABLE 6 Summary of results shown in Examples 1-3. Inhibitor Activator uMInhibitor Construct Construct peptide E_(min)[RLU] EC₅₀[nM] E_(max)[RLU]— C563 50 30,000 10 100,000 — CT139 50 0 20 100,000 C1761 C563 501,000 >10,000 NA C1761 CT139 50 400 >1,000 NA C1759 C563 50 20,000 200100,000 C1760 C563 50 5,000 60 100,000 C1762 C563 50 5,000 600 90,000C2184 C563 50 1,000 >1,000 NA C2183 C563 50 4,000 >1,000 NA C2182 C56350 30,000 30 90,000 C2302 C563 50 0 >10,000 NA C2181 C563 50 0 >10,000NA C2180 C563 50 0 >10,000 NA C2179 C563 50 0 >10,000 NA C2218 C563 501,000 >1,000 NA C2219 C563 50 2,000 >1,000 NA C2220 C563 50 3,000 >1,000NA C2153 C563 50 20,000 2,000 60,000 C2107 CT139 5 0 100 100,000 C2106CT139 5 0 40 100,000

Example 4: TCR-Based Inhibitory Chimeric Antigen Receptors

Construct Design and Cloning

The NY-ESO-1-responsive inhibitory constructs were created using thehigh affinity anti-HLA-A*02:01/NY-ESO-1 1G4α95:LY T cell receptor (TCR)variant. The charged residues in the TM of TCRα (R253 and K258) and TCRβ(K288) were mutated to leucine. Then, the LILRB1 ITIM (residues 484-650)was appended to the mutated TCRα or TCRs. Anti-HLA-A*02:01/MAGE-A3single chain variable fragment (scFv) was generated in-house. Theanti-HLA-A2*02:01/MAGE-A3 chimeric antigen receptor (CAR) used in thisstudy contains the anti-HLA-A*02:01/MAGE-A3 scFv, CD8 hinge, CD28 TM,and CD28, 41BB, and CD3ζ intracellular domains (ICDs). All fragments,including 5′ and 3′ BsmBI sites, were amplified using Q5 polymerase (NewEngland Biolabs) and digested with DpnI (Thermo Scientific) at 37° C.for 60 min. The generated PCR fragments were purified using theNucleospin gel and PCR cleanup kit (Macherey-Nagel). The plasmids wereGolden Gate assembled in a reaction containing BsmBI (ThermoScientific), T4 DNA ligase (Thermo Scientific), 10 mM ATP, and 1×FastDigest buffer (Thermo Scientific).

Jurkat NFAT Activation Assay

Jurkat T lymphocytes that contain firefly luciferase gene under thecontrol of the nuclear factor of activator T cells (NFAT) transcriptionfactor (BPS Bioscience) were co-transfected with plasmids encoding TCRand/or scFv-fusion constructs using the Neon transfection system (ThermoFisher). The electroporated cells were incubated in RPMI mediasupplemented with 20% fetal bovine serum (FBS) heat-inactivated at 56°C. for 60 min (HIA-FBS) and 0.1% pencillin-streptomycin (P/S) (Gibco).TAP deficient T2 lymphoblasts (ATCC RL-1992) were loaded with varyingamounts of a modified NY-ESO-1 peptide (SLLMWITQV) (SEQ ID NO: 107)alone, MAGE-A3 peptide (FLWGPRALV) (SEQ ID NO: 106) alone, or varyingamounts of MAGE-A3 peptide in addition to 50 μM NY-ESO-1 peptide in RPMIsupplemented with 1% BSA and 0.1% P/S (Gibco). Peptides used in theassay were synthesized to >95% purity assessed by mass spectrometry(Genscript). 18 hours post-transfection, Jurkats were resuspended at0.8×10⁶ cells per mL in RPMI supplemented with 10% HIA-FBS and 1% P/S.In a 384 well plate, 12000 Jurkats were co-cultured with 12000peptide-loaded T2s per well at 37° C., 5% CO₂ for 6 hours. NFAT-mediatedluciferase production was measured by adding 15 uL of ONE-Stepluciferase assay reagent (BPS Bioscience). After 20 minutes,luminescence was detected using a plate reader (Tecan).

TCR-Based Inhibitory CARs Using the LILRB1 Intracellular Domain

As shown in Table 7 and FIG. 11, co-expression of TCR-based CARs havingeither only the extracellular domains of the TCR or also having thetransmembrane regions of the TCRs with mutations to polar residues,yield functional inhibitory CARs.

TABLE 7 LBD Hinge TM ICD C563 MAGE-A3 CD8[117-161] CD28[135-161]CD28[136-202] pep1 scFv 41BB[197-238] CD3z[31-127] C2057 TCR LILRB1LILRB1 1G4: α95LY α C2058 TCR LILRB1 LILRB1 1G4: α95LY α C2156 TCR TCRLILRB1 1G4: 1G4: α95LY α α95LY α (R253L/K258L) C2157 TCR TCR LILRB1 1G4:1G4: α95LY β α95LY α (K288L)

TCR-base CARs inhibit the anti-HLA-A*02:01 MAGE-A3 CAR in trans.Jurkat-NFAT luciferase reporter cells were transfected with (1)anti-HLA-A*02:01/MAGE-A3 CAR alone (C563), (2) co-transfected withMAGE-A3 CAR (C563) and TCRα(R253L/K258L)-LILRB1 fusion andTCRβ(K288L)-LILRB1 ICD fusion TCR-inhibitory fusion construct(C2156+C2157) or (3) co-transfected with MAGE-A3 CAR andTCRαECD/TCRβECD-LIRT1(TM+ICD) fusion (C2057+C2058) TCR-inhibitory fusionconstruct. The effects of the two inhibitory variants on NFAT activationwas measured by co-culturing transfected Jurkat cells with T2 cellsloaded with 50 μM NY-ESO-1 peptide in combination with varying amountsof MAGE-A3 peptide. Data are summarized in Table 8.

TABLE 8 Trans effect of TCR-inhibitory fusion constructs on MAGE-A3 CARInhibitor Activator μM Inhibitor Construct Construct peptideE_(min)[RLU] EC₅₀[nM] E_(max)[RLU] — C563 50 19949 24.52 134443 C2057 +C563 50 2072 >10,000 NA C2058 C2156 + C563 50 9198 7108 NA C2157

Truncated TCR alpha or TCR beta extracellular domains (ECD)− with LILRB1TM− ICD fusions inhibit CAR activation. TCR alpha or TCR betaextracellular domains (ECD)− with TCR TM− LILRB1 ICD fusions also actedas inhibitor CARs when the TCR transmembrane mutations abolish TCR-CD3subunit interactions. The experiment demonstrates that the TCR a chainand β chains can be used to generate inhibitory chimeric antigenreceptors by interfering with recruitment of stimulatory factors by CD3subunits.

Example 5: Methods for Examples 6-14

Cell Culture

Jurkat cells encoding an NFAT luciferase reporter were obtained from BPSBioscience. All other cell lines used in this study were obtained fromATCC. In culture, Jurkat cells were maintained in RPMI mediasupplemented with 10% FBS, 1% Pen/Strep and 0.4 mg/mL G418/Geneticin.T2, MCF7, Raji, K562 and HeLa cells were maintained as suggested byATCC. “Normal” Raji cells were made by transducing Raji cells withHLA-A*02 lentivirus (custom lentivirus, Alstem) at a MOI of 5.HLA-A*02-positive Raji cells were sorted using a FACSMelody Cell Sorter(BD).

Plasmid Construction

The NY-ESO-1-responsive inhibitory construct was created by fusing theNY-ESO-1 scFv LBD to domains of receptors including hinge, transmembraneregion, and/or intracellular domain of leukocyte immunoglobulin-likereceptor subfamily B member 1, LILRB1 (LIR-1), programmed cell deathprotein 1, PDCD1 (PD-1), or cytotoxic T-lymphocyte protein 4, CTLA4(CTLA-4). All activating CAR constructs contained an scFv fused to theCD8a hinge, CD28 TM, and CD28, 4-1BB and CD3zeta ICDs. TheCD19-activating CAR scFv was derived from the FMC63 mouse hybridoma.MSLN-activating CAR scFvs were derived from human M5 (LBD1) as describedand humanized SS1 (LBD2). Gene segments were combined using Golden Gatecloning and inserted downstream of a human EF1α promoter contained in alentiviral expression plasmid.

Jurkat Cell Transfection

Jurkat cells were transiently transfected via 100 uL format Neonelectroporation system (Thermo Fisher Scientific) according tomanufacturer's protocol using the following settings: 3 pulses, 1500V,10 msec. Cotransfection was performed with 1-3 ug of activator CAR orTCR construct and 1-3 ug of either scFv or TCR alpha/TCR beta LIR-1blocker constructs or empty vector per 1e6 cells and recovered in RPMImedia supplemented with 20% heat-inactivated FBS and 0.1% Pen/Strep. Toconfirm blocker surface expression, Jurkat cells were stained 18-24hours post-transfection with 10 ug/mL streptavidin-PE-HLA-A*02-pMHCtetramer for 60 minutes at 4° C. in PBS with 1% BSA and characterized byflow cytometry (BD FACSCanto II).

Jurkat-NFAT-Luciferase Activation Studies

Peptides, MAGE-A3 (MP1; FLWGPRALV; SEQ ID NO: 106), MAGE-A3 (MP2;MPKVAELVHFL; SEQ ID NO: 108), HPV E6 (TIHDIILECV; SEQ ID NO: 109), HPVE7 (YMLDLQPET: SEQ ID NO: 110) and modified NY-ESO-1 ESO (ESO;SLLMWITQV; SEQ ID NO: 107), were synthesized by Genscript. Activatingpeptide was serially diluted starting at 50 uM. Blocker peptide,NY-ESO-1, was diluted to 50 uM (unless otherwise indicated) which wasadded to the activating peptide serial dilutions and subsequently loadedonto 1e4 T2 cells in 15 uL of RPMI supplemented with 1% BSA and 0.1%Pen/Strep and incubated in Corning® 384-well Low Flange White FlatBottom Polystyrene TC-treated Microplates. The following day, 1e4 Jurkatcells were resuspended in 15 uL of RPMI supplemented with 10%heat-inactivated FBS and 0.1% Pen/Strep, added to the peptide-loaded T2cells and co-cultured for 6 hours. ONE-Step Luciferase Assay System (BPSBioscience) was used to evaluate Jurkat luminescence. For assaysinvolving high density targets, Jurkat cells were similarly transfectedand cocultured with tumor cells expressing target antigens at variousJurkat:tumor cell ratios. Assays were performed in technical duplicates.

Primary T Cell Transduction, Expansion, and Enrichment

Leukopaks were purchased from AllCells®. Collection protocols and donorinformed consent were approved by an Institutional Review Board (IRB),with strict oversight. HIPAA compliance and approved protocols were alsofollowed. Frozen PBMCs were thawed in 37° C. water bath and cultured at1e6 cells/mL in LymphoONE (Takara) with 1% human serum and activatedusing 1:100 of T cell TransAct (Miltenyi) supplemented with IL-15 (10ng/mL) and IL-21 (10 ng/mL). After 24 hours, lentivirus was added toPBMCs at MOI=5. Activator and blocker receptors were simultaneouslyco-transduced at a MOI=5 for each lentivirus. PBMCs were cultured for2-3 additional days to allow cells to expand under TransAct stimulation.Post expansion, activator and blocker transduced primary T cells wereenriched for blocker-positive T cells by positive selection usinganti-PE microbeads (Miltenyi) according to manufacturer's instructions.Briefly, primary T cells were incubated with 10 ug/mLstreptavidin-PE-HLA-A*02-pMHC tetramer for 60 minutes at 4° C. in MACSbuffer (0.5% BSA+2 mM EDTA in PBS). Cells were washed 3 times in MACSbuffer and passed through the LS column (Miltenyi) to separateblocker-positive cells (a mix of blocker-only and activator+blockercells) from untransduced and activator-only cells.

Primary T Cell In Vitro Cytotoxicity Studies

For cytotoxicity studies with pMHC targets, enriched primary T cellswere incubated with 2e3 MCF7 cells expressing Renilla luciferase(Biosettia) loaded with a titration of target peptide as described aboveat an effector:target ratio of 3:1 for 48 hours. Liveluciferase-expressing MCF7 cells were quantified using a RenillaLuciferase Reporter Assay System (Promega). For cytotoxicity studieswith non-pMHC targets, enriched primary T cells were incubated with 2e3WT Raji cells (“tumor” cells) or HLA-A*02 transduced Raji cells(“normal” cells) at an effector:target ratio of 3:1 for up to 6 days. WT“tumor” Raji cells stably expressing GFP and Renilla luciferase(Biosettia) or HLA-A*02 transduced “normal” Raji cells stably expressingRFP and firefly luciferase (Biosettia) were imaged together withunlabeled primary T cells using an IncuCyte live cell imager.Fluorescence intensity of live Raji cells over time was quantified usingIncuCyte imaging software. For reversibility studies, enriched primary Tcells were similarly cocultured with “normal” or “tumor” Raji cells for3 days and imaged. After 3 days, T cells were separated from remainingRaji cells using CD19 negative selection and reseeded with fresh“normal” or “tumor” Raji cells as described. In separate wells, liveluciferase-expressing Raji cells were quantified using a Dual-LuciferaseReporter Assay System (Promega) at 72 hours. For studies in which IFNγsecretion was assessed, supernatants collected after 48 hours ofco-culture were tested for IFNγ using a BD Human IFNγ flex kit followingmanufacturer's exact instructions.

Mouse Xenograft Study

Frozen PBMCs were thawed in 37° C. water bath and rested overnight inserum-free TexMACS Medium (Miltenyi) prior to activation. PBMCs wereactivated in 1.5e6 cells/mL using T cell TransAct (Miltenyi) and TexMACSMedium supplemented with IL-15 (20 ng/mL) and IL-21 (20 ng/mL). After 24hours, lentivirus was added to PBMCs at a MOI of 5. PBMCs were culturedfor 8-9 additional days to allow cells to expand under TransActstimulation. Post expansion, T cells were enriched on A2-LIR-1 usinganti-PE microbeads (Miltenyi) against streptavidin-PE-HLA-A*02-pMHC for2-5 additional days prior to in vivo injection. Enriched T cells werealso validated by flow cytometry (BD FACSCanto II) for expression ofCD19 scFv activator and HLA-A*02 LIR-1 blocker by sequential stainingwith CD19-Fc (1:100; R&D Systems) and goat anti-human IgG-FITC (1:200;Invitrogen) for activator and 10 ug/mL streptavidin-APC-HLA-A*02-pMHCfor blocker.

In vivo experiments were conducted by Explora BioLabs underInstitutional Animal Care and Use Committee (IACUC)-approved protocols.5-6 week old female NOD.Cg-Prkdcscid Il2rgtm1WjlTg(HLA-A/H2-D/B2M)1Dvs/SzJ (NSG-HLA-A2/HHD) mice were purchased from TheJackson Labs. Animals were acclimated to the housing environment for atleast 3 days prior to the initiation of the study. Animals were injectedwith 2e6 WT Raji cells or HLA-A*02 transduced Raji cells in 100 uLvolume subcutaneously in the right flank. When tumors reached an averageof 70 mm3 (V=L×W×W/2), animals were randomized into 5 groups (n=7) and2e6 or 1e7 T cells were administered via the tail vein. Post T cellinjection, tumor measurements were performed 3 times per week and bloodwas collected 10 days and 17 days after for flow analysis. One animalfrom the WT Raji group receiving 1e7 CD19-CAR+A2-LIR-1 T cells wasexcluded from the study due to a failed tail vein injection, followed byflow cytometry confirmation of the absence of human T cells in theblood. At each time point, human T cells in the blood were quantified byflow cytometry (BD FACSCanto II) post RBC lysis. Cells were stained withanti-mouse CD45-FITC (clone 30-F11), anti-human CD3-PE (clone SK7),anti-human CD4-APC (clone OKT4), and anti-human CD8-PerCP-Cy5.5 (cloneRPA-T8). All antibodies were obtained from Biolegend and used at a 1:100dilution. DAPI (Invitrogen) was used to exclude dead cells fromanalysis. For histopathological analysis, tumor samples were fixed,sectioned and stained for huCD3 (clone EP449E). Image quantification wasdone using ImageJ software.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism software. Allpeptide and cell titration studies are shown as mean±standard deviation(SD), while in vitro and in vivo studies using primary T cells are shownas mean±standard error of the mean (SEM), unless otherwise noted.Peptide and cell titration curves were fit using a four-parameternon-linear regression analysis. EC50 values were calculated directlyfrom the curves. All other groups of data were analyzed using anordinary two-way ANOVA followed by a Tukey's multiple-comparisons test,unless otherwise noted.

Example 6: Assaying the Effect of the LIR-1 Hinge on Blocking Activity

The effects of different LIR-1 hinges on the ability of HLA-A*02 scFvLIR-1 inhibitory receptors to block killing by Jurkat cells expressing aKRAS TCR activator was assayed using the Jurkat NFat Luciferase assaysdescribed supra. A humanized PA2.1 scFv LIR-1 receptor and humanizedBB7.2 scFv LIR-1 with a shorter LIR-1 hinge were assayed in Jurkat cellsas previously described, and the results are shown in FIGS. 12A-12B.Jurkat cells were transfected with a KRAS TCR activator receptor and/orHLA-A*02 scFv LIR-1 inhibitory receptor (humanized PA2.1 or humanizedBB7.2) with a variety of LIR-1 derived hinges, and co-cultured with T2target cells that were either HLA:A11 positive, or HA:A11 and HLA:A02positive. Inhibitory receptors with both the shorter and longer hingebehaved similarly (FIG. 12A-12B). An inhibitory receptor with a mousePA2.1 scFv and slightly longer hinges was also assayed functionedsimilarly to shorter LIR-1 hinges in the T2-Jurkat assay (FIG. 13A-13B).Hinge sequences are shown in black in FIG. 12B and FIG. 13B, with thegray SS in the sequences in FIG. 13B representing a linker between theantigen-binding domain and the hinge, and the gray VIGIL the start ofthe LLR-1 transmembrane domain. Hinge, transmembrane domain andintracellular domain of the inhibitory receptors were all derived fromLIR-1. FIGS. 12A-12B and 13A-13B show that LIR-1 hinge length can bevaried without negatively effecting the LIR-1 inhibitory receptor.Shorter hinges can provide an advantage when packaging nucleic acidsequences encoding LIR-1 inhibitory receptors in lentiviral vectors fordelivery.

TABLE 9 Sequences of constructs Amino Acid Nucleotide Name SequenceSequence PA TEFTLTISSLQPD GACATTCAAATGACC 2.1.14 DFATYYCFQGSHVCAGAGCCCATCCACC (VL:VH) PRTFGQGTKVEVK CTGAGCGCATCTGTA scFv GGGGSGGGGSGGGGGTGACCGGGTCACC LIR1 GSGGQVQLVQSGA ATCACTTGTAGATCC sHTICD EVKKPGSSVKVSCAGTCAGAGTATTGTA KASGYTFTSYHIH CACAGTAATGGGAAC WVRQAPGQGLEWIACCTATTTGGAATGG GWIYPGNVNTEYN TATCAGCAGAAACCA EKFKGKATITADEGGTAAAGCCCCAAAA STNTAYMELSSLR TTGCTCATCTACAAA SEDTAVYYCAREEGTCTCTAACAGATTT ITYAMDYWGQGTL AGTGGTGTACCAGCC VTVSSVVSGPSGGAGGTTCAGCGGTTCC PSSPTTGPTSTSG GGAAGTGGTACTGAA PEDQPLTPTGSDPTTCACCCTCACGATC QSGLGRHLGVVIG TCCTCTCTCCAGCCA ILVAVILLLLLLLGATGATTTCGCCACT LLFLILRHRRQGK TATTACTGTTTTCAA HWTSTQRKADFQHGGTTCACATGTGCCG PAGAVGPEPTDRG CGCACATTCGGTCAG LQWRSSPAADAQEGGTACTAAAGTAGAA ENLYAAVKHTQPE GTCAAAGGCGGAGGT DGVEMDTRSPHDEGGAAGCGGAGGGGGA DPOAVTYAEVKHS GGATCTGGCGGCGGA RPRREMASPPSPLGGAAGCGGAGGGCAG SGEFLDTKDRQAE GTGCAGCTGGTGCAG EDRQMDTEAAASETCTGGGGCTGAGGTG APQDVTYAQLHSL AAGAAGCCTGGGTCC TLRREATEPPPSQTCAGTGAAGGTTTCC EGPSPAVPSIYAT TGCAAGGCTTCTGGA LAIH (SEQ IDTACACCTTCACTAGC NO: 92) TATCATATACATTGG GTGCGCCAGGCCCCC GGACAAGGGCTTGAGTGGATCGGATGGATC TACCCTGGCAATGTT AACACAGAATATAAT GAGAAGTTCAAGGGCAAAGCCACCATTACC GCGGACGAATCCACG AACACAGCCTACATG GAGCTGAGCAGCCTGAGATCTGAAGACACG GCTGTGTATTACTGT GCGAGGGAGGAAATT ACCTACGCTATGGACTACTGGGGCCAGGGA ACCCTGGTCACCGTG TCCTCAGTGGTCTCA GGAGCGTCTGGGGGCCCCAGCTCCCCGACA ACAGGCCCCACCTCC ACATCTGGCCCTGAG GACCAGCCCCTCACCCCCACCGGGTCGGAT CCCCAGAGTGGTCTG GGAAGGCACCTGGGG GTTGTGATCGGCATCTTGGTGGCCGTCATC CTACTGCTCCTCCTC CTCCTCCTCCTCTTC CTCATCCTCCGAGATCGACGTCAGGGCAAA CACTGGACATCGACC CAGAGAAAGGCTGAT TTCCAACATCCTGCAGGGGCTGTGGGGCCA GAGCCCACAGACAGA GGCCTGCAGTGGAGG TCCAGCCCAGCTGCCGATGCCCAGGAAGAA AACCTCTATGCTGCC GTGAAGCACACACAG CCTGAGGATGGGGTGGAGATGGACACTCGG AGCCCACACGATGAA GACCCCCAGGCAGTG ACGTATGCCGAGGTGAAACACTCCAGACCT AGGAGAGAAATGGCC TCTCCTCCTTCCCCA CTGTCTGGGGAATTCCTGGACACAAAGGAC AGACAGGCGGAAGAG GACAGGCAGATGGAC ACTGAGGCTGCTGCATCTGAAGCCCCCCAG GATGTGACCTACGCC CAGCTGCACAGCTTG ACCCTCAGACGGGAGGCAACTGAGCCTCCT CCATCCCAGGAAGGG CCCTCTCCAGCTGTG CCCAGCATCTACGCCACTCTGGCCATCCAC TAG (SEQ ID NO: 114) PA DIQMTQSPSTLSA GACATTCAAATGACC2.1.14 SVGDRVTITCRSS CAGAGCCCATCCACC (VL:VH) QSIVHSNGNTYLECTGAGCGCATCTGTA scFv WYQQKPGKAPKLL GGTGACCGGGTCACC LIR1 IYKVSWRFSGVPAATCACTTGTAGATCC HTICD RFSGSGSGTEFTL AGTCAGAGTATTGTA TISSLQPDDFATYCACAGTAATGGGAAC YCFQGSHVPRTFG ACCTATTTGGAATGG QGTKVEVKGGGGSTATCAGCAGAAACCA GGGGSGGGGSGGQ GGTAAAGCCCCAAAA VQLVQSGAEVKKPTTGCTCATCTACAAA GSSVKVSCKASGY GTCTCTAACAGATTT TFTSYHIHWVRQAAGTGGTGTACCAGCC PGQGLEWIGWIYP AGGTTCAGCGGTTCC GNVNTEYNEKFKGGGAAGTGGTACTGAA KATITADESTNTA TTGACCCTCACGATC YMELSSLRSEDTATCCTCTCTCCAGCCA VYYCAREEITYAM GATGATTTCGCCACT DYWGQGTLVTVSSTATTACTGTTTTCAA YGSQSSKPYLLTH GGTTCACATGTGCCG PSDPLELVVSGPSCGCACATTCGGTCAG GGPSSPTTGPTST GGTACTAAAGTAGAA SGPEDQPLTPTGSGTCAAAGGCGGAGGT DPQSGLGRHLGVV GGAAGCGGAGGGGGA IGILVAVILLL1LGGATCTGGCGGCGGA LLLLHLILRHRRQ GGAAGCGGAGGCCAG GKHWTSTQRKADFGTGCAGCTGGTGCAG QHPAGAVGPEPTD TCTGGGGCTGAGGTG RGLQWRSSPAADAAAGAAGGCTGGGTCC QEENLYAAVKHTQ TCAGTGAAGGTTTCC PEDGVEMDTRSPHTGCAAGGCTTCTGGA DEDPQAVTYAEVK TACACCTTCACTAGC HSRPRREMASPPSTATCATATACATTGG PLSGEFLDTKDRQ GTGCGCCAGGCCCCC AEEDRQMDTEAAAGGACAAGGGCTTGAG SEAPQDVTYAQLH TGGATCGGATGGATC SLTLRRSATEPPPTACCCTGGCAATGTT SQEGPSPAVPSIY AACACAGAATATAAT ATLAIH(SEQ GAGAAGTTCAAGGGCID NO: 91) AAAGCCACCATTACC GCGGACGAATCCACG AACACAGCCTACATGGAGCTGAGCAGCCTG AGATCTGAAGACACG GCTGTGTATTACTGT GCGAGGGAGGAAATTACCTACGCTATGGAC TACTGGGGCCAGGGA ACCCTGGTCACCGTG TCCTCATACGGCTCACAGAGCTCCAAACCC TACCTGCTGACTCAC CCCAGTGACCCCCTG GAGCTCGTGGTCTCAGGACCGTCTGGGGGC CCCAGCTCCCCGACA ACAGGCCCCACCTCC ACATCTGGCCCTGAGGACCAGCCCCTCACC CCCACCGGGTCGGAT CCCCAGAGTGGTCTG GGAAGGCACCTGGGGGTTGTGATCGGCATC TTGGTGGCCGTCATC CTACTGCTCCTCCTC CTCCTCCTCCTCTTCCTCATCCTCCGACAT CGACGTCAGGGCAAA CACTGGACATCGACC CAGAGAAAGGCTGATTTCCAACATCCTGCA GGGGCTGTGGGGCCA GAGCCCACAGACAGA GGCCTGCAGTGGAGGTCCAGCCCAGCTGCC GATGCCCAGGAAGAA AACCTCTATGCTGCC GTGAAGCACACACAGCCTGAGGATGGGGTG GAGATGGACACTCGG AGCCCACACGATGAA GACCCCCAGGCAGTGACGTATGCCGAGGTG AAACACTCCAGACCT AGGAGAGAAATGGCC TCTCCTCCTTCCCCACTGTCTGGGGAATTC CTGGACACAAAGGAC AGACAGGCGGAAGAG GACAGGCAGATGGACACTGAGGCTGCTGCA TCTGAAGCCCCCCAG GATGTGACCTACGCC CAGCTGCACAGCTTGACCCTCAGACGGGAG GCAACTGAGCCTCCT CCATCCCAGGAAGGG CCCTCTCCAGCTGTGCCCAGCATCTACGCC ACTCTGGCCATCCAC TAG (SEQ ID NO: 113) PA QVQLVQSGAEVKKCAGGTGCAGCTGGTG 2.1.14 PGSSVKVSCKASG CAGTCTGGGGCTGAG scFv YTFTSYHIHWVRQGTGAAGAAGCCTGGG LIR1 APGQGLEWIGWIY TCCTCAGTGAAGGTT sHTICD PGNVNTEYNEKFKTCCTGCAAGGCTTCT GKATITADESTNT GGATACACCTTCACT AYMELSSLRSEDTAGCTATCATATACAT AVYYCAREEITYA TGGGTGCGCCAGGCC MDYWGQGTLVTVSCCCGGACAAGGGCTT SGGGGSGGGGSGG GAGTGGATCGGATGG GGSGGDIQMTQSPATCTACCCTGGCAAT STLSASVGDRVTI GTTAACACAGAATAT TCRSSQSIVHSNGAATGAGAAGTTCAAG NTYLEWYQQKPGK GGCAAAGCCACCATT APKLLIYKVSNRFACCGCGGACGAATCC SGVPARFSGSGSG ACGAACACAGCCTAC TEFTLTISSLQPDATGGAGCTGAGCAGC DFATYYCFQGSHV CTGAGATCTGAAGAC PRTFGQGTKVEVKACGGCTGTGTATTAC VVSGPSGGPSSPT TGTGCGAGGGAGGAA TGPTSTSGPEDQPATTACCTACGCTATG LTPTGSDPQSGLG GACTACTGGGGCCAG RHLGVVIGILVAVGGAACCCTGGTCACC ILLLLLLLLLFLI GTGTCCTCAGGCGGA LRHRRQGKHWTSTGGTGGAAGCGGAGGG QRKADFQHPAGAV GGAGGATCTGGCGGC GPEPTDRGLQWRSGGAGGAAGCGGAGGC SPAADAQEENLYA GACATTCAAATGACC AVKHTQPEDGVEMCAGAGCCCATCCACC DTRSPHDEDPQAV CTGAGCGCATCTGTA TYAEVKHSRPRREGGTGACCGGGTCACC MASPPSPLSGEFL ATCACTTGTAGATCC DTKDRQAEEDRQMAGTCAGAGTATTGTA DTEAAASEAPQDV CACAGTAATGGGAAC TYAQLHSLTLRREACCTATTTGGAATGG ATEPPPSQEGPSP TATCAGCAGAAACCA AVPSIYATLAIHGGTAAAGCCCCAAAA (SEQ ID NO: TTGCTCATCTACAAA 90) GTCTCTAACAGATTTAGTGGTGTACCAGCC AGGTTCAGCGGTTCC GGAAGTGGTACTGAA TTCACCCTCACGATCTCCTCTCTCCAGCCA GATGATTTCGCCACT TATTACTGTTTTCAA GGTTCACATGTGCCGCGCACATTCGGTCAG GGTACTAAAGTAGAA GTCAAAGTGGTCTCA GGACCGTCTGGGGGCCCCAGCTCCCCGACA ACAGGCCCCACCTCC ACATCTGGCCCTGAG GACCAGCCCCTCACCCCCACCGGGTCGGAT CCCCAGAGTGGTCTG GGAAGGCACCTGGGG GTTGTGATCGGCATCTTGGTGGCCGTCATC CTACTGCTCCTCCTC CTCCTCCTCCTCTTC CTCATCCTCCGACATCGACGTCAGGGCAAA CACTGGACATCGACC CAGAGAAAGGCTGAT TTCCAACATCCTGCAGGGGCTGTGGGGCCA GAGCCCACAGACAGA GGCCTGCAGTGGAGG TCCAGCCCAGCTGCCGATGCCCAGGAAGAA AACCTCTATGCTGCC GTGAAGCACACACAG CCTGAGGATGGGGTGGAGATGGACACTCGG AGCCCACACGATGAA GACCCCCAGGCAGTG ACGTATGCCGAGGTGAAACACTCCAGACCT AGGAGAGAAATGGCC TCTCCTCCTTCCCCA CTGTCTGGGGAATTCCTGGACACAAAGGAC AGACAGGCGGAAGAG GACAGGCAGATGGAC ACTGAGGCTGCTGCATCTGAAGCCCCCCAG GATGTGACCTACGCC CAGCTGCACAGCTTG ACCCTCAGACGGGAGGCAACTGAGCCTCCT CCATCCCAGGAAGGG CCCTCTCCAGCTGTG CCCAGCATCTACGCCACTCTGGCCATCCAC TAG (SEQ ID NO: 112) PA2.1.1 QVQLVQSGAEVKKCAGGTGCAGCTGGTG 4 scFv PGSSVKVSCKASG CAGTCTGGGGCTGAG LIR1 YTFTSYHIHWVRQGTGAAGAAGCCTGGG HTICD APGQGLSWIGWIY TCCTCAGTGAAGGTT PGNVNTEYNEKFKTCCTGCAAGGCTTCT GKATITADESTNT GGATACACCTTCACT AYMELSSLRSEDTAGCTATCATATACAT AVYYCAREEITYA TGGGTGCGCCAGGCC MDYWGQGTLVTVSCCCGGACAAGGGCTT SGGGGSGGGGSGG GAGTGGATCGGATGG GGSGGDIQMTQSPATCTACCCTGGCAAT STLSASVGDRVTI GTTAACACAGAATAT TCRSSQSIVHSNGAATGAGAAGTTCAAG NTYLEWYQQKPGK GGCAAAGCCACCATT APKLLIYKVSNRFACCGCGGACGAATCC SGVFARFSGSGSG ACGAACACAGCCTAC TEFTLTISSLQPDATGGAGCTGAGCAGC DFATYYCFQGSHV CTGAGATCTGAAGAC PRTFGQGTKVEVKACGGCTGTGTATTAC YGSQSSKPYLLTH TGTGCGAGGGAGGAA PSDPLELVVSGPSATTACCTACGCTATG GGPSSPTTGPTST GACTACTGGGGCCAG SGPEDQPLTPTGSGGAACCCTGGTCACC DPQSGLGRHLGVV GTGTCCTCAGGCGGA IGILVAVILLLLLGGTGGAAGCGGAGGG LLLLFLILRHRRQ GGAGGATCTGGCGGC GKHWTSTQRKADFGGAGGAAGCGGAGGC QHPAGAVGPEPTD GACATTCAAATGACC RGLQWRSSPAADACAGAGCCCATCCACC QEENLYAAVKHTQ CTGAGCGCATCTGTA PEDGVEMDTRSPHGGTGACCGGGTCACC DEDPQAVTYAEVK ATCACTTGTAGATCC HSRPRREMASPPSAGTCAGAGTATTGTA PLSGEFLDTKDRQ CACAGTAATGGGAAG AEEDRQMDTEAAAACCTATTTGGAATGG SEAPQDVTYAQLH TATCAGCAGAAACCA SLTLRREATEPPPGGTAAAGCCCCAAAA SQEGPSPAVPSIY TTGCTCATCTACAAA ATLAIH (SEQGTCTCTAACAGATTT ID NO: 89) AGTGGTGTACCAGCC AGGTTCAGCGGTTCCGGAAGTGGTACTGAA TTCACCCTCACGATC TCCTCTCTCCAGCCA GATGATTTCGCCACTTATTACTGTTTTCAA GGTTCACATGTGCCG CGCACATTCGGTCAG GGTACTAAAGTAGAAGTCAAATACGGCTCA CAGAGCTCCAAACCC TACCTGCTGACTCAC CCCAGTGACCCCCTGGAGCTCGTGGTCTCA GGACCGTCTGGGGGC CCCAGCTCCCCGACA ACAGGCCCCACCTCCACATCTGGCCCTGAG GACCAGCCCCTCACC CCCACCGGGTCGGAT CCCCAGAGTGGTCTGGGAAGGCACCTGGGG GTTGTGATCGGCATC TTGGTGGCCGTCATC CTACTGCTCCTCCTCCTCCTCCTCCTCTTC CTCATCCTCCGACAT CGACGTCAGGGCAAA CACTGGACATCGACCCAGAGAAAGGCTGAT TTCCAACATCCTGCA GGGGCTGTGGGGCCA GAGCCCACAGACAGAGGCCTGCAGTGGAGG TCCAGCCCAGCTGCC GATGCCCAGGAAGAA AACCTCTATGCTGCCGTGAAGCACACACAG CCTGAGGATGGGGTG GAGATGGACACTCGG AGCCCACACGATGAAGACCCCCAGGCAGTG ACGTATGCCGAGGTG AAACACTCCAGACCT AGGAGAGAAATGGCCTCTCCTCCTTCCCCA CTGTCTGGGGAATTC CTGGACACAAAG GACAGACAGGCGGAAGAGGACAGGCAGATG GACACTGAGGCTGCT GCATCTGAAGCCCCC CAGGATGTGACCTACGCCCAGCTGCACAGC TTGACCCTCAGACGG GAGGCAACTGAGCCT CCTCCATCCCAGGAAGGGCCCTCTCCAGCT GTGCCCAGCATCTAC GCCACTCTGGCCATC CACTAG (SEQ ID NO: 111)

Example 7: Comparison of LIR-1, CTLA-4 and PD-1 Inhibitory Receptors

An NY-ESO-1-responsive inhibitory construct was created by fusing theNY-ESO-1 scFv LBD to domains of receptors including hinge, transmembraneregion, and/or intracellular domain of leukocyte immunoglobulin-likereceptor subfamily B member 1, LILRB1 (LIR-1), programmed cell deathprotein 1, PDCD1 (PD-1), or cytotoxic T-lymphocyte protein 4, CTLA4(CTLA-4). MAGE-A3 activating CAR constructs contained an scFv fused tothe CD8a hinge, CD28 TM, and CD28, 4-1BB and CD 3zeta intracellulardomains (ICDs). Gene segments were combined using Golden Gate cloningand inserted downstream of a human EF1α promoter contained in alentiviral expression plasmid.

Initially, peptide-MHC (pMHC) targets for both the activator and blockerreceptors were used, because pMHCs allow convenient quantification ofthe pharmacology of the system (FIG. 14A). Specifically, a single-chainfragment variable (scFv) that binds HLA-A*02-NY-ESO-1_((SLLMWITQC/V))was used as the inhibitor receptor ligand-binding domain (LBD), and asecond scFv against HLA-A*02-MA GE-A3_((FLWGPRALV)) pMHC (Gallo,unpublished) was as part of an activator receptor third-generation CAR.Jurkat effector cells that express luciferase upon NFAT activation wereused to readout activator sensitivity, with EC50 values reporting thehalf-maximum activator peptide concentration required for a response.Both PD-1 and CTLA-4 intracellular domains (ICD) mediated a shift inEC50 of activation in Jurkat cells of less than ˜10×, measured bytitration of peptides loaded on T2 cells as stimulus (FIG. 14B).

A variety of potential inhibitor (blocker) receptor constructs werescreened, and the LIR-1 blocker constructed was discovered to havestronger blocking properties than PD-1 and CTLA-4. This blocker receptorincludes the intracellular, transmembrane (TM) and hinge domains of theLIR-1 (LILRB1) receptor, one of several LIR-family molecules encoded bythe human genome. The LIR-1 blocker (henceforth referred to as LIR-1)fused to the NY-ESO-1 LBD mediated an EC50 shift of >5,000× (FIG. 14B,FIGS. 17A-17D). A control, titration of unrelated HLA-A*02-bindingpeptides provided an estimate of the shift caused by competition ofloaded peptides on T2 cells for available HLA molecules, a contributionto the total shift typically less than ˜10× (FIG. 15). For the EC50shift values reported here, comparisons were typically to EC50s ofactivator-only constructs. Further, for a given pair ofactivator/blocker receptors, the mid-point of titration for inhibitionwas approximately constant and depended on the ratio of activating toblocking peptide, presumably directly correlated with the target-antigenratio (FIGS. 16A-16D). The target concentration explored in the majorityof these experiments is estimated to have ranged from ˜1,000-10,000copies/cell.

Example 8: LIR-1 Inhibitory Receptors with Multiple scFv Ligand BindingDomains

The activity of the LIR-1 inhibitory receptor was tested with a varietyof antigen-binding domains specific to other pMHC targets. For fourdifferent pMHC targets, a total of six different scFvs grafted onto theLIR-1 mediated dramatic shifts in EC50, ranging from 10 to 1,000× (FIG.14C). With respect to its interaction with activator receptors, theLIR-1 receptor was also robust; its blocking behavior was applicable tomultiple targets and scFvs (FIG. 14D). The blockade was ligand-dependent(FIG. 17A), although many LIR-1 constructs produced lower basal/tonicsignaling when paired with specific activator receptors. The EC50 shiftsdepended on the presence of a fused ICD, as LIR-1 constructs completelylacking ICD, or containing mutations in key elements of the ICD, had noeffect (FIG. 17B). The ligand-independent blocker activity, however, hadlittle effect on the activation EC50 absent ligand (FIG. 16A-16D). LIR-1inhibitory receptors are a modular, adaptable, ligand-gated system thatfunctions across multiple targets and antigen-binding domains.

Example 9: LIR-1 Inhibitory Domains Fused to TCR Alpha and TCR Beta

The LIR-1 inhibitory receptor was tested when fused to TCRalpha and TCRbeta subunits, or when in combination with a TCR activator receptor.TCRs directed against 3 different pMHC targets, 2 from MAGE-A3 and onefrom HPV (see Methods, supra). In every case, LIR-1 shifted theactivation EC50 by large amounts, estimated to range >1,000× (FIG. 14E;FIG. 19A). Furthermore, an NY-ESO-1 TCR LBD, when fused to LIR-1, alsoproduced substantial EC50 shifts (FIG. 14F; FIG. 19B). Indeed, allcombinations of a MAGE-A3_((FLWGPRALV)) CAR or TCR with anNY-ESO-1_((SLLMWITQV)) scFv or TCR displayed large shifts. Thus, theLIR-1 inhibitory receptor exhibited modularity that encompassed bothCARs and TCRs.

Example 10: LIR-1 Inhibitory Receptors Respond to Target Antigen Presentin Cis to Activator Receptor Target Antigen

The ability of the LIR-1 blocker receptor to inhibit activation by anactivator receptor when activator and inhibitor targets were presentedin cis was assayed. In a first assay, a simplified stimulus consistingof target-loaded beads roughly the size of cells (d˜2.8 um) was used.Jurkat cells expressing activator and blocker receptors were activatedonly by beads that contained the A (activator) target, not by beads withdual A/B (activator/blocker) targets (FIG. 18A). Interestingly, theeffector cells were activated by a mixture of A+ and B+ beads, even whenthe A+ beads comprised only 20% of the total. This demonstrated that thecells expressing the activator and blocker receptors are: (i) blockedfrom activation by the blocker receptor when the targets are present incis on the same surface; and, (ii) activated by individual A+ beadsamong an excess of B+ beads.

Example 11: LIR-1 Inhibitory Receptors and Cell Surface Antigens

The ability of the LIR-1 inhibitor receptor to block activation inresponse to non-pMHC targets, representing surface antigens that canextend into the realm of 100,000 epitopes/cell, was assayed. scFvs thatbind either the B-cell marker CD19, the solid-tumor antigen mesothelin(MSLN), or HLA-A*02 in a peptide-independent fashion were tested. Inthese cases, the target antigen concentration was not controlled, aswith exogenous peptide as with pMHCs. Instead, the ratio of activator toblocker expression was varied using different DNA concentrations intransient transfection assays. Though assay sensitivity preventedexploration of the full range of EC50 shifts, shifts in Emax over 10×were observed. These experiments showed that the properties of the LIR-1receptor in a dual receptor system were generally the same forhigh-density targets (FIG. 18B; FIGS. 16-16D, FIG. 21A) and that theblocker receptor blocked activation of the A receptor to an extentreflected by its relative surface level on the effector cells (FIG.18C). The LIR-1 receptor also behaved in a modular manner in thishigh-antigen-density setting. An scFv against HLA-A*02 acted either asactivator (FIG. 18D) or blocker when fused to either an activator orblocker receptor, respectively. The LIR-1 receptor is flexible enough toaccommodate low and high target densities, in principle allowingoptimization for pMHC targets as well as non-pMHC surface antigens.

Example 12: LIR-1 Inhibitory Receptors in Primary T Cells

The ability of the LIR-1 receptor to block activation of primary T cellswas assayed. pMHC targets were used initially. After enrichment fortransduced T cells via physical selection, engineered T cells expressingactivator and blocker receptors were assayed using target cellsengineered to express luciferase as the readout for viable cells. Withan HPV TCR as activator, the NY-ESO-1 scFv fused to LIR-1 shifted thecell-count vs. peptide-concentration curve in peptide-loaded MCF7 tumorcells by ˜25× (FIG. 20A; FIG. 23). Thus, the behavior of LIR-1 receptordemonstrated in Jurkat cell activation assays was extended to primary Tcell functions, including cytotoxicity.

To establish proof of concept, the HLA-A*02 LIR-1 construct was shown tofunction as a blocker in the presence of pMHC-dependent activators inJurkat cells with T2 target cells (FIG. 20B). For the activator, a CD19scFv was used as a CAR. This activator/blocker pair was shown tofunction together as robustly as other pairs previously tested in Jurkatcells (FIG. 18B). T model tumor cells that differentially express theLIR-1 receptor ligand, CD19-positive, HLA-A*02-negative Raji cells wereused. To model the corresponding normal cells, the same cell line stablyexpressing the HLA-A*02 gene was used (FIG. 24A). The cell linesactivated Jurkat cells if the target cells expressed CD19 only, but notif they expressed both CD19 and HLA-A*02: i.e., the HLA-A*02 blockerreceptor blocked activation by CD19-CAR in a ligand-dependent way (FIG.24B).

The CD19/HLA-A*02 receptor pair also worked in primary T cells (FIG.21). Engineered T cells killed CD19-expressing Raji cells in the absenceof HLA-A*02 expression. Raji cells that expressed both CD19 and HLA-A*02were blocked from cytotoxicity. Importantly, primary T cells bearing thereceptor pairs distinguished CD19+ “tumor” from CD19+/HLA-A*02+ “normal”cells in a mixed culture. These findings mirrored results from theabove-mentioned bead experiment, but in a more complex cellular settingwhere the effector cells' cytotoxicity focused on “tumor” targets, evenwhen surrounded by “normal” cells. The degree of selectivity wasimpressive given that neither receptor had been subjected to deliberateoptimization for maximal selectivity. This behavior was confirmed with asecond antigen, MSLN (FIG. 22B).

Example 13: Reversibility of Inhibition by LIR-1 Inhibitory Receptors

The LIR-1 inhibitory receptor was tested for its ability to functionreversibly; that is, to cycle from a state of blockade to activation andback to blockade. Effector T cells expressing the LIR-1 receptor andactivator receptor were tested to see if they could function reversiblyand iteratively. The effector cells were co-cultured with Raji cells,either to mimic tumor (CD19+) or normal (CD19+/HLA-A*02+) cellencounter. After each round of exposure to target cells, the Raji cellswere removed from the culture and a new population of target cells wasintroduced. Cytotoxicity and gamma-interferon (IFNγ) were measured atthe end of each round. In both permutations, block-kill-block andkill-block-kill, the T cells functioned as required by a celltherapeutic of this type (FIGS. 25A-25D). They reversibly cycled from astate of block to cytotoxicity and back, depending on the target cellsto which they were exposed. This result demonstrates that a T cellexpressing an activator receptor and the LIR-1 inhibitory receptor donot get stuck in one state (blockade or activation), but rather canswitch back and forth as it integrates signals from normal and tumorcells. Additionally, these experiments were reproduced in primary Tcells from multiple donors (FIGS. 21A-D, FIGS. 25A-25D; FIGS. 26A-26B),despite their complexity, heterogeneity and donor-to-donor variability,demonstrating the robust functions of the LIR-1 inhibitory receptor.

Example 14: Human T Cells Expressing an Activator Receptor and LIR-1Blocker Receptor Selectively Target Cancer Cells in a Mouse Model

The ability of the CD19/HLA-A*02 activator/blocker pair engineered inprimary T cells to allow expansion of the T cells in vitro to largenumbers using standard CD3/CD28 stimulation was assayed (FIG. 27A; FIG.28A). Thus, T cells expressing the activator and LIR-1 receptors c canbe scaled up to sufficient numbers for animal experiments and ultimatelyfor patients.

The CD19/HLA-A*02 activator/blocker combination was tested in vitro todemonstrate selective killing of CD19+ tumor cells, while sparingCD19+/HLA-A*02+ cells in a mouse xenograft cancer model (FIG. 27B). Thesame two Raji cell lines described above, one CD19+ and the otherCD19+/HLA-A*02+, were injected into the flanks of immunocompromised(NGS-HLA-A2.1) mice. T cells engineered with the test constructs wereinjected at two doses, 2e6 (not shown) or 1e7 T cells. The growth of thetumor, as well as the persistence of the implanted T cells, wereanalyzed overtime. Only the CD19+ tumor cells were killed in the mouseand the tumor control tracked with transferred T cell numbers, promotingsurvival of the host mice (FIGS. 27C-27E, FIGS. 28B-28C). TheCD19+/HLA-A*02+ cells designed to model normal cells were unaffected.Human CD4+/CD8+ cell ratios in the blood of mice bearing engineeredcells tracked with their control counterpart; i.e., in “tumor”-graftedmice, CD4+>CD8+ cells, as in the CD19 CAR positive control mice; and, in“normal”-grafted mice, CD4+<CD8+ cells, similar to mice harboringuntransduced T cells (FIG. 28D). Moreover, huCD3+ cells in the tumor(FIGS. 29A-29B) correlated inversely with tumor volume, and engineered Tcells expressing the two receptors behaved like untransduced T cells in“normal” grafts (low infiltrate), and like the CD19 CAR in “tumor”grafts (high infiltrate). Finally, the mice appeared normal with regardto typical clinical observations (Table 10). Together, these resultsdemonstrated that the LIR-1 inhibitory receptor can function hi vivo toinhibit an activator receptor, in a simplified setting devised to mimicthe different normal and tumor cell types that will be encountered inpatients.

TABLE 10 Clinical observations of mice during in vivo experiment StudyDay: 6 8 10 13 15 17 20 22 24 28 29 Group Animal ID CO CO CO CO CO CO COCO CO CO CO Tumor 59 N N N N N N N N N N N, EUT Untreated 100 N N N N NN N N N N N 61 N N N N N N N N N 19AN(1) 19AN(1) 285 N N N N N N N N N NN, EUT 289 N N N N N N N N 19AN(1) N N, EUT 87 N N N N N N N N N N N,EUT 60 N N N N N N N N N N N Tumor 286 N N N N N N N N N 19AN(1)19AN(1), EUT Untransduced 78 N N N N N N N N N N N 76 N N N N N N N N NN N 89 N N N N N N N N N N N, EUT 90 N N N N N N N N N N N 275 N N N N NN N N N N N 83 N N N N N N N N N 19AN(1) 19AN(1), EUT Tumor 280 N N N NN 19AN(1) 19AN(1) 19AN(1) 19AN(1) N N CD19-CAR 99 N N N N N N N N N N Nonly 284 N N N N N N N N N N N 71 N N N N N N N N N N N 279 N N N N N NN N N N N 77 N N N N N N N N N N N 94 N N N N N N N N N N N Tumor 64 N NN N N N N N N N N CD19-CAR + 69 N N N N N N N N N N N A2-LIR1 274 N N NN N N N N N N N 96 N N N N N N N N 19AN(1) 19AN(1) 19AN(1) 277 N N N N NN N N 19AN(1) 19AN(1) 19AN(1) 97 N N N N N N N N N N N “Normal” 66 N N NN N N N N 19AN(1) 19AN(2) 19AN(2), EUT Untreated 294 N N N N N N N19AN(1) 19AN(1) 19AN(2) 19AN(2), EUT 293 N N N N N N N 19AN(1) 19AN(1)19AN(2) 19AN(2), EUT 91 N N N N N N N 19AN(1) 19AN(1) 19AN(2) 19AN(2),EUT 287 N N N N N N N N 19AN(1) 19AN(2) 19AN(2), EUT 281 N N N N N N N19AN(1) 19AN(1) 19AN(2) 19AN(2), EUT 282 N N N N N N N N 19AN(1) 19AN(1)19AN(1), EUT “Normal” 290 N N N N N N N N N 19AN(1) 19AN(1), EUTUntransduced 25 N N N N N N N N 19AN(1) 19AN(1) 19AN(1), EUT 93 N N N NN N N N N N N, EUT 84 N N N N N N N N N N N, EUT 292 N N N N N N N N19AN(1) 19AN(1) 19AN(1) 65 N N N N N N N N N 19AN(1) 19AN(1), EUT 79 N NN N N N N N N N N “Normal” 270 N N N N N N N N N N N CD19-CAR 283 N N NN N N N N N N N only 82 N N N N N N N N N N N 92 N N N N N 19AN(1)19AN(1) 19AN(1) 19AN(1) 19AN(1) 19AN(1) 95 N N N N N N N 19AN(1) 19AN(1)19AN(1) 19AN(1) 80 N N N N N N N N N N N 297 N N N N N N N 19AN(1)19AN(1) 19AN(1) 19AN(1) “Normal” 86 N N N N N N N N N N N CD19-CAR + 74N N N N N N N N N N N A2-LIR1 288 N N N N N N N N N N N 273 N N N N N NN N N N N 70 N N N N N N N N 19AN(1) 19AN(1) 19AN(1) 73 N N N N N N N N19AN(1) 19AN(1) 19AN(1) 72 N N N N N N N N 19AN(1) 19AN(1) 19AN(1), EUT

Tumor and “normal” cells were injected on study day 0 and treatmentstarted on study day 10. Clinical observation (CO) were performed3×/week focusing on poor health, stress and pain. Per IACUC guidelines,mice were euthanized if tumors reached >2000 mm³. Codes: N=normal;19A=Abnormal Tumor [N=Necrotic, O=Open], EUT=Euthanized. Severity codes:0=Not present, 1=Moderate, 2=Severe.

What is claimed is:
 1. A chimeric antigen receptor comprising apolypeptide comprising: a) an antigen binding domain; b) an LILRB1 hingedomain or functional fragment or variant thereof; c) a transmembranedomain; and d) an LILRB1 intracellular domain comprising at least oneimmunoreceptor tyrosine-based inhibitory motif (ITIM) selected from thegroup consisting of NLYAAV (SEQ ID NO: 8), VTYAEV (SEQ ID NO: 9), VTYAQL(SEQ ID NO: 10), and SIYATL (SEQ ID NO: 11).
 2. The receptor of claim 1,wherein the intracellular domain comprises a sequence at least 95%identical to SEQ ID NO:
 7. 3. The receptor of claim 1, wherein thetransmembrane domain comprises a LILRB1 transmembrane domain or afunctional fragment or variant thereof.
 4. The receptor of claim 2,wherein the LILRB1 transmembrane domain or functional fragment orvariant thereof comprises a sequence at least 95% identical to SEQ IDNO:
 5. 5. The receptor of claim 1, wherein the LILRB1 hinge domain orfunctional fragment or variant thereof comprises a sequence at least 95%identical to SEQ ID NO: 4, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 80,SEQ ID NO:81, SEQ ID NO: 82, SEQ ID NO: 83 or SEQ ID NO:
 84. 6. Thereceptor of claim 1, wherein the LILRB1 hinge and transmembrane comprisea sequence at least 95% identical to SEQ ID NO:
 20. 7. The receptor ofclaim 1, wherein the LILRB1 transmembrane and intracellular domaincomprise a sequence at least 95% identical to SEQ ID NO:
 21. 8. Thereceptor of claim 1, wherein the LILRB1 hinge, transmembrane andintracellular domains comprise a sequence at least 95% identical to SEQID NO:
 3. 9. The receptor of claim 1, wherein the LILRB1 hinge,transmembrane and intracellular domains comprise a sequence at least 95%identical to SEQ ID NO:
 2. 10. The receptor of claim 1, wherein theLILRB1 hinge, transmembrane and intracellular domains comprise SEQ IDNO:
 2. 11. The receptor of claim 1, wherein the antigen binding domainis specific to an antigen that is lost in a cancer cell through loss ofheterozygosity.
 12. The receptor of claim 1, wherein the antigen bindingdomain is specific to a minor histocompatibility antigen (MiHA).
 13. Thereceptor of claim 1, wherein the antigen binding domain is specific toan antigen that is lost in a cancer cell through loss of Y chromosome.14. The receptor of claim 1, wherein the antigen binding domain isspecific to a major histocompatibility class I allele.
 15. The receptorof claim 14, wherein the major histocompatibility class I allelecomprises HLA-A, HLA-B, HLA-C allele or HLA-E allele.
 16. The receptorof claim 15, wherein the HLA-A allele is HLA-A*02.
 17. The receptor ofclaim 1, wherein the antigen binding domain comprises a single chainvariable fragment (scFv).
 18. The receptor of claim 17, wherein the scFvcomprises complementarity determined regions (CDRs) selected from thegroup consisting of SEQ ID NOS: 22-33.
 19. The receptor of claim 17,wherein the scFv comprises a sequence at least 95% identical to any oneof SEQ ID NOS: 35-46 or
 125. 20. The receptor of claim 1, wherein thereceptor comprises an amino acid sequence at least 95% identical to anyone of SEQ ID NOS: 48-50, 52-53, 55-56, 59-65, 67-68, 77, 121 or 122.21. The receptor of claim 1, wherein the receptor comprises an aminoacid sequence of SEQ ID NOS: 48-50, 52-53, 55-56, 59-65, 67-68, 77, 121or
 122. 22. A polynucleotide comprising a nucleic acid sequence encodingthe polypeptide of claim
 1. 23. A vector comprising the polynucleotideof claim
 22. 24. An immune cell comprising the receptor of claim
 1. 25.The immune cell of claim 24, wherein the immune cell is a T cell. 26.The immune cell of 25, further comprising an activator receptor.
 27. Amethod of treating a subject with a disease or a disorder, comprisingadministering to the subject the immune cell of claim 26.